Monkeypox virus quadrivalent mRNA vaccine induces immune response and protects against vaccinia virus.

Monkeypox has been declared a public health emergency by the World Health Organization. There is an urgent need for efficient and safe vaccines against the monkeypox virus (MPXV) in response to the rapidly spreading monkeypox epidemic. In the age of COVID-19, mRNA vaccines have been highly successful and emerged as platforms enabling rapid development and large-scale preparation. Here, we develop two MPXV quadrivalent mRNA vaccines, named mRNA-A-LNP and mRNA-B-LNP, based on two intracellular mature virus specific proteins (A29L and M1R) and two extracellular enveloped virus specific proteins (A35R and B6R). By administering mRNA-A-LNP and mRNA-B-LNP intramuscularly twice, mice induce MPXV specific IgG antibodies and potent vaccinia virus (VACV) specific neutralizing antibodies. Further, it elicits efficient MPXV specific Th-1 biased cellular immunity, as well as durable effector memory T and germinal center B cell responses in mice. In addition, two doses of mRNA-A-LNP and mRNA-B-LNP are protective against the VACV challenge in mice. And, the passive transfer of sera from mRNA-A-LNP and mRNA-B-LNP-immunized mice protects nude mice against the VACV challenge. Overall, our results demonstrate that mRNA-A-LNP and mRNA-B-LNP appear to be safe and effective vaccine candidates against monkeypox epidemics, as well as against outbreaks caused by other orthopoxviruses, including the smallpox virus.

Introduction of Multilayered Dual-Signal Nanotags into a Colorimetric-Fluorescent Coenhanced Immunochromatographic Assay for Ultrasensitive and Flexible Monitoring of SARS-CoV-2.

Timely, accurate, and rapid diagnosis of SARS-CoV-2 is a key factor in controlling the spread of the epidemic and guiding treatments. Herein, a flexible and ultrasensitive immunochromatographic assay (ICA) was proposed based on a colorimetric/fluorescent dual-signal enhancement strategy. We first fabricated a highly stable dual-signal nanocomposite (SADQD) by continuously coating one layer of 20 nm AuNPs and two layers of quantum dots onto a 200 nm SiO2 nanosphere to provide strong colorimetric signals and enhanced fluorescence signals. Two kinds of SADQD with red and green fluorescence were conjugated with spike (S) antibody and nucleocapsid (N) antibody, respectively, and used as dual-fluorescence/colorimetric tags for the simultaneous detection of S and N proteins on one test line of ICA strip, which can not only greatly reduce the background interference and improve the detection accuracy but also achieve a higher colorimetric sensitivity. The detection limits of the method for target antigens via colorimetric and fluorescence modes were as low as 50 and 2.2 pg/mL, respectively, which were 5 and 113 times more sensitive than those from the standard AuNP-ICA strips, respectively. This biosensor will provide a more accurate and convenient way to diagnose COVID-19 in different application scenarios.

CESSAT: A chemical additive-enhanced single-step accurate CRISPR/Cas13 testing system for field-deployable ultrasensitive detection and genotyping of SARS-CoV-2 variants of concern.

The continued emergence of SARS-CoV-2 variants of concern (VOCs) has raised great challenges for epidemic prevention and control. A rapid, sensitive, and on-site SARS-CoV-2 genotyping technique is urgently needed for individual diagnosis and routine surveillance. Here, a field-deployable ultrasensitive CRISPR-based diagnostics system, called Chemical additive-Enhanced Single-Step Accurate CRISPR/Cas13 Testing system (CESSAT), for simultaneous screening of SARS-CoV-2 and its five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) within 40 min was reported. In this system, a single-step reverse transcription recombinase polymerase amplification-CRISPR/Cas13a assay was incorporated with optimized extraction-free viral lysis and reagent lyophilization, which could eliminate complicated sample processing steps and rigorous reagent storage conditions. Remarkably, 10% glycine as a chemical additive could improve the assay sensitivity by 10 times, making the limit of detection as low as 1 copy/µL (5 copies/reaction). A compact optic fiber-integrated smartphone-based device was developed for sample lysis, assay incubation, fluorescence imaging, and result interpretation. CESSAT could specifically differentiate the synthetic pseudovirus of SARS-CoV-2 and its five VOCs. The genotyping results for 40 clinical samples were in 100% concordance with standard method. We believe this simple but efficient enhancement strategy can be widely incorporated with existing Cas13a-based assays, thus leading a substantial progress in the development and application of rapid, ultrasensitive, and accurate nucleic acid analysis technology.

Introduction of graphene oxide-supported multilayer-quantum dots nanofilm into multiplex lateral flow immunoassay: A rapid and ultrasensitive point-of-care testing technique for multiple respiratory viruses.

A lateral flow immunoassay (LFA) biosensor that allows the sensitive and accurate identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other common respiratory viruses remains highly desired in the face of the coronavirus disease 2019 pandemic. Here, we propose a multiplex LFA method for the on-site, rapid, and highly sensitive screening of multiple respiratory viruses, using a multilayered film-like fluorescent tag as the performance enhancement and signal amplification tool. This film-like three-dimensional (3D) tag was prepared through the layer-by-layer assembly of highly photostable CdSe@ZnS-COOH quantum dots (QDs) onto the surfaces of monolayer graphene oxide nanosheets, which can provide larger reaction interfaces and specific active surface areas, higher QD loads, and better luminescence and dispersibility than traditional spherical fluorescent microspheres for LFA applications. The constructed fluorescent LFA biosensor can simultaneously and sensitively quantify SARS-CoV-2, influenza A virus, and human adenovirus with low detection limits (8 pg/mL, 488 copies/mL, and 471 copies/mL), short assay time (15 min), good reproducibility, and high accuracy. Moreover, our proposed assay has great potential for the early diagnosis of respiratory virus infections given its robustness when validated in real saliva samples. Electronic Supplementary Material: Supplementary material (Section S1 Experimental section, Section S2 Calculation of the maximum number of QDs on the GO@TQD nanofilm, Section S3 Optimization of the LFA method, and Figs. S1-S17 mentioned in the main text) is available in the online version of this article at 10.1007/s12274-022-5043-6.

Neutralization sensitivity, fusogenicity, and infectivity of Omicron subvariants.

BACKGROUND: The emergence of SARS-CoV-2 Omicron subvariants has raised questions regarding resistance to immunity by natural infection or immunization. We examined the sensitivity of Delta and Omicron subvariants (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/5, and BA.3) to neutralizing antibodies from BBIBP-CorV-vaccinated and BBIBP-CorV- or ZF2001-boosted individuals, as well as individuals with Delta and BA.1 breakthrough infections, and determined their fusogenicity and infectivity. METHODS: In this cross-sectional study, serum samples from two doses of BBIBP-CorV-vaccinated individuals 1 (n = 36), 3 (n = 36), and 7 (n = 37) months after the second dose; BBIBP-CorV- (n = 25) or ZF2001-boosted (n = 30) individuals; and fully vaccinated individuals with Delta (n = 30) or BA.1 (n = 26) infection were collected. The serum-neutralizing reactivity and potency of bebtelovimab were assessed against D614G, Delta, and Omicron subvariants (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/5, and BA.3) through a pseudovirus neutralization assay. The fusogenicity and infectivity of D614G, Delta, and Omicron subvariants were determined by cell-cell fusion assay and pseudovirus infection assay, respectively. RESULTS: Omicron subvariants markedly escaped vaccine-elicited neutralizing antibodies after two doses of BBIBP-CorV with comparable efficiency. A third dose vaccination of BBIBP-CorV or ZF2001 increased neutralizing antibody titers and breadth against Delta and three Omicron subvariants. Delta and BA.1 breakthrough infections induced comparable neutralizing antibody titers against D614G and Delta variants, whereas BA.1 breakthrough infections elicited a stronger and broader antibody response against three Omicron subvariants than Delta breakthrough infections. BA.2.12.1 and BA.4/5 are more resistant to immunity induced by breakthrough infections. Bebtelovimab had no significant loss of potency against the Delta and Omicron subvariants. Cell culture experiments showed Omicron subvariants to be less fusogenic and have higher infectivity than D614G and Delta with comparable efficiency. CONCLUSIONS: These findings have important public health implications and highlight the importance of repeated exposure to SARS-CoV-2 antigens to broaden the neutralizing antibody response against Omicron subvariants.

Microfluidic chip and isothermal amplification technologies for the detection of pathogenic nucleic acid.

The frequency of outbreaks of newly emerging infectious diseases has increased in recent years. The coronavirus disease 2019 (COVID-19) outbreak in late 2019 has caused a global pandemic, seriously endangering human health and social stability. Rapid detection of infectious disease pathogens is a key prerequisite for the early screening of cases and the reduction in transmission risk. Fluorescence quantitative polymerase chain reaction (qPCR) is currently the most commonly used pathogen detection method, but this method has high requirements in terms of operating staff, instrumentation, venues, and so forth. As a result, its application in the settings such as poorly conditioned communities and grassroots has been limited, and the detection needs of the first-line field cannot be met. The development of point-of-care testing (POCT) technology is of great practical significance for preventing and controlling infectious diseases. Isothermal amplification technology has advantages such as mild reaction conditions and low instrument dependence. It has a promising prospect in the development of POCT, combined with the advantages of high integration and portability of microfluidic chip technology. This study summarized the principles of several representative isothermal amplification techniques, as well as their advantages and disadvantages. Particularly, it reviewed the research progress on microfluidic chip-based recombinase polymerase isothermal amplification technology and highlighted future prospects.

Recombinase Polymerase Amplification Combined with Fluorescence Immunochromatography Assay for On-Site and Ultrasensitive Detection of SARS-CoV-2.

This study established a portable and ultrasensitive detection method based on recombinase polymerase amplification (RPA) combined with high-sensitivity multilayer quantum dot (MQD)-based immunochromatographic assay (ICA) to detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The RPA-MQD-based ICA method is reported for the first time and has the following advantages: (i) RPA is free from the constraints of instruments and can be promoted in point-of-care testing (POCT) scenarios, (ii) fluorescence ICA enhances the portability of detection operation so that the entire operation time is controlled within 1 h, and (iii) compared with common colorimetric-based RPA-ICA, the proposed assay used MQD to provide strong and quantifiable fluorescence signal, thus enhancing the detection sensitivity. With this strategy, the proposed RPA-MQD-based ICA can amplify and detect the SARS-CoV-2 nucleic acid on-site with a sensitivity of 2 copies/reaction, which is comparable to the sensitivity of commercial reverse transcription quantitative polymerase chain reaction (RT-qPCR) kits. Moreover, the designed primers did not cross-react with other common respiratory viruses, including adenovirus, influenza virus A, and influenza virus B, suggesting high specificity. Thus, the established portable method can sensitively detect SARS-CoV-2 nucleic acid without relying on equipment, having good application prospects in SARS-CoV-2 detection scenarios under non-lab conditions.

Introduction of graphene oxide-supported multilayer-quantum dots nanofilm into multiplex lateral flow immunoassay: A rapid and ultrasensitive point-of-care testing technique for multiple respiratory viruses

A lateral flow immunoassay (LFA) biosensor that allows the sensitive and accurate identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other common respiratory viruses remains highly desired in the face of the coronavirus disease 2019 pandemic. Here, we propose a multiplex LFA method for the on-site, rapid, and highly sensitive screening of multiple respiratory viruses, using a multilayered film-like fluorescent tag as the performance enhancement and signal amplification tool. This film-like three-dimensional (3D) tag was prepared through the layer-by-layer assembly of highly photostable CdSe@ZnS−COOH quantum dots (QDs) onto the surfaces of monolayer graphene oxide nanosheets, which can provide larger reaction interfaces and specific active surface areas, higher QD loads, and better luminescence and dispersibility than traditional spherical fluorescent microspheres for LFA applications. The constructed fluorescent LFA biosensor can simultaneously and sensitively quantify SARS-CoV-2, influenza A virus, and human adenovirus with low detection limits (8 pg/mL, 488 copies/mL, and 471 copies/mL), short assay time (15 min), good reproducibility, and high accuracy. Moreover, our proposed assay has great potential for the early diagnosis of respiratory virus infections given its robustness when validated in real saliva samples. Electronic Supplementary Material Supplementary material (Section S1 Experimental section, Section S2 Calculation of the maximum number of QDs on the GO@TQD nanofilm, Section S3 Optimization of the LFA method, and Figs. S1–S17 mentioned in the main text) is available in the online version of this article at 10.1007/s12274-022-5043-6.

Individual heterogeneity and airborne infection: Effect of non-uniform air distribution

The classical Wells–Riley equation assumes homogeneity of susceptible individuals and environments to airborne exposure. However, individual susceptibility to infection is mostly heterogeneous, and exposure variability could arise from differences in inhalation rate, spatiotemporal non-uniformity of infectious aerosol concentrations, and the exposure trajectory and time. Non-uniform air distribution results in spatial non-uniformity of infectious aerosol concentrations. The non-uniformity effect is essentially a problem of individual infection probability. Here, we derived a general dose-response equation and a heterogeneous Wells–Riley equation accounting for individual variability in infection probability. The heterogeneous Wells-Riley equation shows the potential of the zone air distribution effectiveness to consider spatial non-uniformity under steady-state conditions. An existing quanta generation rate formula was theoretically justified. The new equation was then applied to a restaurant reporting an outbreak of coronavirus disease 2019, with spatial and/or temporal heterogeneity of infectious aerosol concentrations. Our results show the need to include spatial non-uniformity in outbreak investigations. A hypothetical two-zone setup was used to demonstrate how the inter-zonal distribution of clean air and the inter-zonal exchange flow affect airborne infections. An infector in a poorly diluted zone with the greatest number of susceptible individuals would result in the most secondary infections, whereas an infector in a well-ventilated zone with few susceptible individuals would result in the least secondary infections.

Highly sensitive and rapid detection of SARS-CoV-2 via a portable CRISPR-Cas13a-based lateral flow assay.

To rapidly identify individuals infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and control the spread of coronavirus disease (COVID-19), there is an urgent need for highly sensitive on-site virus detection methods. A clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)-based molecular diagnostic method was developed for this purpose. Here, a CRISPR system-mediated lateral flow assay (LFA) for SARS-CoV-2 was established based on multienzyme isothermal rapid amplification, CRISPR-Cas13a nuclease, and LFA. To improve the limit of detection (LoD), the crispr RNA, amplification primer, and probe were screened, in addition to concentrations of various components in the reaction system. The LoD of CRISPR detection was improved to 0.25 copy/µl in both fluorescence- and immunochromatography-based assays. To enhance the quality control of the CRISPR-based LFA method, glyceraldehyde-3-phosphate dehydrogenase was detected as a reference using a triple-line strip design in a lateral flow strip. In total, 52 COVID-19-positive and 101 COVID-19-negative clinical samples examined by reverse transcription polymerase chain reaction (RT-PCR) were tested using the CRISPR immunochromatographic detection technique. Results revealed 100% consistency, indicating the comparable effectiveness of our method to that of RT-PCR. In conclusion, this approach significantly improves the sensitivity and reliability of CRISPR-mediated LFA and provides a crucial tool for on-site detection of SARS-CoV-2.

Tetra-primer ARMS-PCR combined with dual-color fluorescent lateral flow assay for the discrimination of SARS-CoV-2 and its mutations with a handheld wireless reader.

Several virulent variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged along with the spread of this virus throughout the population. Some variants can exhibit increased transmissibility and reduced immune neutralization reactivity. These changes are deeply concerning issues that may hinder the ongoing effort of epidemic control measures, especially mass vaccination campaigns. The accurate discrimination of SARS-CoV-2 and its emerging variants is essential to contain the coronavirus disease 2019 pandemic. Herein, we report a low-cost, facile, and highly sensitive diagnostic platform that can simultaneously distinguish wild-type (WT) SARS-CoV-2 and its two mutations, namely, D614G and N501Y, within 2 h. WT or mutant (M) nucleic acid fragments at each allelic locus were selectively amplified by the tetra-primer amplification refractory mutation system (ARMS)-PCR assay. Allele-specific amplicons were simultaneously detected by two test lines on a quantum dot nanobead (QB)-based dual-color fluorescent test strip, which could be interpreted by the naked eye or by a home-made fluorescent strip readout device that was wirelessly connected to a smartphone for quantitative data analysis and result presentation. The WT and M viruses were indicated and were strictly discriminated by the presence of a green or red band on test line 1 for the D614G site and test line 2 for the N501Y site. The limits of detection (LODs) for the WT and M D614G were estimated as 78.91 and 33.53 copies per µL, respectively. This assay was also modified for the simultaneous detection of the N and ORF1ab genes of SARS-CoV-2 with LODs of 1.90 and 6.07 copies per µL, respectively. The proposed platform can provide a simple, accurate, and affordable diagnostic approach for the screening of SARS-CoV-2 and its variants of concern even in resource-limited settings.

A rapid water bath PCR combined with lateral flow assay for the simultaneous detection of SARS-CoV-2 and influenza B virus† † Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ra07756b

The outbreak of the coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global health and economic threats to the human society. Thus, a rapid and accurate detection method for early testing and diagnosis should be established. In this study, a rapid water bath polymerase chain reaction (PCR) combined with lateral flow assay was developed to detect SARS-CoV-2 and influenza B virus simultaneously. A homemade automated transfer device equipped with reaction tube shuttled rapidly between two water baths at 98 °C and 53 °C to realize rapid PCR. After amplification, two-ended labeled PCR products were detected using the lateral flow strip with two test lines and streptavidin-conjugated quantum dot nanobeads. The fluorescence value was read using a handheld instrument. The established assay could complete reverse-transcription PCR amplification and lateral flow detection in 45 minutes. The detection limits were 8.44 copies per μL and 14.23 copies per μL for SARS-CoV-2 and influenza B virus, respectively. The coefficients of variation of the test strip were 10.10% for the SARS-CoV-2 and 4.94% for the influenza B virus, demonstrating the excellent repeatability of the experiment. These results indicated that the rapid PCR combined with lateral flow assay could detect SARS-CoV-2 and influenza B virus simultaneously at a short assay time and low cost, thereby showing the remarkable potential for the rapid and multiplex detection of respiratory viruses in resource-limited settings. Rapid and highly sensitive multiplex detection of SARS-CoV-2 and influenza B virus using water bath PCR-combined fluorescent lateral flow assay.

A rapid water bath PCR combined with lateral flow assay for the simultaneous detection of SARS-CoV-2 and influenza B virus.

The outbreak of the coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global health and economic threats to the human society. Thus, a rapid and accurate detection method for early testing and diagnosis should be established. In this study, a rapid water bath polymerase chain reaction (PCR) combined with lateral flow assay was developed to detect SARS-CoV-2 and influenza B virus simultaneously. A homemade automated transfer device equipped with reaction tube shuttled rapidly between two water baths at 98 °C and 53 °C to realize rapid PCR. After amplification, two-ended labeled PCR products were detected using the lateral flow strip with two test lines and streptavidin-conjugated quantum dot nanobeads. The fluorescence value was read using a handheld instrument. The established assay could complete reverse-transcription PCR amplification and lateral flow detection in 45 minutes. The detection limits were 8.44 copies per µL and 14.23 copies per µL for SARS-CoV-2 and influenza B virus, respectively. The coefficients of variation of the test strip were 10.10% for the SARS-CoV-2 and 4.94% for the influenza B virus, demonstrating the excellent repeatability of the experiment. These results indicated that the rapid PCR combined with lateral flow assay could detect SARS-CoV-2 and influenza B virus simultaneously at a short assay time and low cost, thereby showing the remarkable potential for the rapid and multiplex detection of respiratory viruses in resource-limited settings.

Rational preparation and application of a mRNA delivery system with cytidinyl/cationic lipid.

The messenger RNA (mRNA)-based therapy, especially mRNA vaccines, has shown its superiorities in versatile design, rapid development and scale production, since the outbreak of coronavirus disease 2019 (COVID-19). Although the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines had been approved for application, unexpected adverse events were reported to be most likely associated with the mRNA delivery systems. Thus, the development of mRNA delivery system with good efficacy and safety remains a challenge. Here, for the first time, we report that the neutral cytidinyl lipid, 2-(4-amino-2-oxopyrimidin-1-yl)-N-(2,3-dioleoyl-oxypropyl) acetamide (DNCA), and the cationic lipid, dioleoyl-3,3'-disulfanediylbis-[2-(2,6-diaminohexanamido)] propanoate (CLD), could encapsulate and deliver the COVID-19 mRNA-1096 into the cytoplasm to induce robust adaptive immune response. In the formulation, the molar ratio of DNCA/CLD to a single nucleotide of COVID-19 mRNA-1096 was about 0.9: 0.5: 1 (the N/P ratio was about 7: 1). The DNCA/CLD-mRNA-1096 lipoplexes were rationally prepared by the combination of the lipids DNCA/CLD with the aqueous mRNA solution under mild sonication to stimulate multiple interactions, including H-bonding, π-stacking and electrostatic force between the lipids and the mRNA. After intramuscular applications of the DNCA/CLD-mRNA-1096 lipoplexes, robust neutralizing antibodies and long-lived Th1-biased SARS-CoV-2-specific cell immunity were detected in the immunized mice, thus suggesting the DNCA/CLD a promising mRNA delivery system. Moreover, our study might also inspire better ideas for developing mRNA delivery systems.

Inhibitory effect on SARS-CoV-2 infection of neferine by blocking Ca2+ -dependent membrane fusion.

The coronavirus disease 2019 (COVID-19) pandemic has focused attention on the need to develop effective therapeutics against the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and also against other pathogenic coronaviruses. In this study, we report on a kind of bisbenzylisoquinoline alkaloid, neferine, as a pan-coronavirus entry inhibitor. Neferine effectively protected HEK293/hACE2 and HuH7 cell lines from infection by different coronaviruses pseudovirus particles (SARS-CoV-2, SARS-CoV-2 [D614G, N501Y/D614G, 501Y.V1, 501Y.V2, 501Y.V3 variants], SARS-CoV, MERS-CoV) in vitro, with median effect concentration (EC50 ) of 0.13-0.41 µM. Neferine blocked host calcium channels, thus inhibiting Ca2+ -dependent membrane fusion and suppressing virus entry. This study provides experimental data to support the fact that neferine may be a promising lead for pan-coronaviruses therapeutic drug development.

Self-assembled polymeric micelle as a novel mRNA delivery carrier.

mRNA-based therapy has been evaluated in preclinical and clinical studies for the treatment of a wide variety of disease such as cancer immunotherapies and infectious disease vaccines. However, it remains challenging to development safe and efficient delivery system. Here, we have designed a novel self-assembled polymeric micelle based on vitamin E succinate modified polyethyleneimine copolymer (PVES) to delivery mRNA. In vitro, PVES could transfect mRNA into multiple cell lines such as HEK-293T, HeLa and Vero and the transfection efficiencies were much higher than PEI 25 k. In addition, the cytotoxicity of PVES was much lower than PEI 25 k. Furthermore, mice administered intramuscularly with PVES/SARS-CoV-2 mRNA vaccine induced potent antibody response and show no obvious toxicity. These results demonstrated the potential of PVES as a safe and effective delivery carrier for mRNA.

Ultrasensitive and Simultaneous Detection of Two Specific SARS-CoV-2 Antigens in Human Specimens Using Direct/Enrichment Dual-Mode Fluorescence Lateral Flow Immunoassay.

Sensitive point-of-care methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in clinical specimens are urgently needed to achieve rapid screening of viral infection. We developed a magnetic quantum dot-based dual-mode lateral flow immunoassay (LFIA) biosensor for the high-sensitivity simultaneous detection of SARS-CoV-2 spike (S) and nucleocapsid protein (NP) antigens, which is beneficial for improving the detection accuracy and efficiency of SARS-CoV-2 infection in the point-of-care testing area. A high-performance magnetic quantum dot with a triple-QD shell (MagTQD) nanotag was first fabricated and integrated into the LFIA system to provide superior fluorescence signals, enrichment ability, and detectability for S/NP antigen testing. Two detection modes were provided by the proposed MagTQD-LFIA. The direct mode was used for rapid screening or urgent detection of suspected samples within 10 min, and the enrichment mode was used for the highly sensitive and quantitative analysis of SARS-CoV-2 antigens in biological samples without the interference of the "hook effect." The simultaneous detection of SARS-CoV-2 S/NP antigens was conducted in one LFIA strip, and the detection limits for two antigens under direct and enrichment modes were 1 and 0.5 pg/mL, respectively. The MagTQD-LFIA showed high accuracy, specificity, and stability in saliva and nasal swab samples and is an efficient tool with flexibility to meet the testing requirements for SARS-CoV-2 antigens in various situations.