Hand-powered centrifugal micropipette-tip with distance-based quantification for on-site testing of SARS-CoV-2 virus.

This paper proposed a hand-powered centrifugal micropipette-tip strategy, termed HCM, for all-in-one immunoassay combined with a distance-based readout for portable quantitative detection of SARS-CoV-2. The target SARS-CoV-2 virus antigen triggers the binding of multiple monoclonal antibody-coated red latex nanobeads, forming larger complexes. Following incubation and centrifugation, the formed aggregated complexes settle at the bottom of the tip, while free red nanobeads remain suspended in the solution. The HCM enables sensitive (1 ng/mL) and reliable quantification of SARS-CoV-2 within 25 min. With the advantages of free washing, free fabrication, free instrument, and without the optical device, the proposed low-cost and easy-to-use HCM immunoassay shows great potential for quantitative POC diagnostics for SARS-CoV-2.

Nanopore membrane chip-based isolation method for metabolomic analysis of plasma small extracellular vesicles from COVID-19 survivors.

Multiple studies showed that metabolic disorders play a critical role in respiratory infectious diseases, including COVID-19. Metabolites contained in small extracellular vesicles (sEVs) are different from those in plasma at the acute stage, while the metabolic features of plasma sEVs of COVID-19 survivors remain unknown. Here, we used a nanopore membrane-based microfluidic chip for plasma sEVs separation, termed ExoSEC, and compared the sEVs obtained by UC, REG, and ExoSEC in terms the time, cost, purity, and metabolic features. The results indicated the ExoSEC was much less costly, provided higher purity by particles/proteins ratio, and achieved 205-fold and 2-fold higher sEVs yield, than UC and REG, respectively. Moreover, more metabolites were identified and several signaling pathways were significantly enriched in ExoSEC-sEVs compared to UC-sEVs and REG-sEVs. Furthermore, we detected 306 metabolites in plasma sEVs using ExoSEC from recovered asymptomatic (RA), moderate (RM), and severe/critical COVID-19 (RS) patients without underlying diseases 3 months after discharge. Our study demonstrated that COVID-19 survivors, especially RS, experienced significant metabolic alteration and the dysregulated pathways mainly involved fatty acid biosynthesis, phenylalanine metabolism, etc. Metabolites of the fatty acid biosynthesis pathway bore a significantly negative association with red blood cell counts and hemoglobin, which might be ascribed to hypoxia or respiratory failure in RM and RS but not in RA at the acute stage. Our study confirmed that ExoSEC could provide a practical and economical alternative for high throughput sEVs metabolomic study.

Merging microfluidics with luminescence immunoassays for urgent point-of-care diagnostics of COVID-19.

The Coronavirus disease 2019 (COVID-19) outbreak has urged the establishment of a global-wide rapid diagnostic system. Current widely-used tests for COVID-19 include nucleic acid assays, immunoassays, and radiological imaging. Immunoassays play an irreplaceable role in rapidly diagnosing COVID-19 and monitoring the patients for the assessment of their severity, risks of the immune storm, and prediction of treatment outcomes. Despite of the enormous needs for immunoassays, the widespread use of traditional immunoassay platforms is still limited by high cost and low automation, which are currently not suitable for point-of-care tests (POCTs). Microfluidic chips with the features of low consumption, high throughput, and integration, provide the potential to enable immunoassays for POCTs, especially in remote areas. Meanwhile, luminescence detection can be merged with immunoassays on microfluidic platforms for their good performance in quantification, sensitivity, and specificity. This review introduces both homogenous and heterogenous luminescence immunoassays with various microfluidic platforms. We also summarize the strengths and weaknesses of the categorized methods, highlighting their recent typical progress. Additionally, different microfluidic platforms are described for comparison. The latest advances in combining luminescence immunoassays with microfluidic platforms for POCTs of COVID-19 are further explained with antigens, antibodies, and related cytokines. Finally, challenges and future perspectives were discussed.

G-triplex: A new type of CRISPR-Cas12a reporter enabling highly sensitive nucleic acid detection.

CRISPR-Cas12a (Cpf1) trans-cleaves ssDNA and this feature has been widely harnessed for nucleic acid detection. Herein, we introduce a new type of Cas12a reporter, G-triplex (G3), and a highly sensitive biosensor termed G-CRISPR. We proved that Cas12a trans-cleaves G3 structures in about 10 min and G3 can serve as an excellent reporter based on the cleavage-induced high-order structure disruption. G3 reporter improves the analytical sensitivity up to 20 folds, enabling the detection of unamplified and amplified DNA as low as 50 pmol and 0.1 amol (one copy/reaction), respectively. G-CRISPR has been utilized for the analysis of 27 PCR-amplified patient samples with HPV infection risk based on both fluorescence and lateral flow assays, resulting in 100% concordance between the two. In comparison with the clinical results, it achieved overall specificity and sensitivity of 100% and 94.7%, respectively. These results suggest that G-CRISPR can serve as a rapid, sensitive, and reliable biosensor, and could further expand the CRISPR toolbox in biomedical diagnostics.

Development and multicenter performance evaluation of fully automated SARS-CoV-2 IgM and IgG immunoassays.

Objectives: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread globally. The laboratory diagnosis of SARS-CoV-2 infection has relied on nucleic acid testing; however, it has some limitations, such as low throughput and high rates of false negatives. Tests of higher sensitivity are needed to effectively identify infected patients. Methods: This study has developed fully automated chemiluminescent immunoassays to determine IgM and IgG antibodies to SARS-CoV-2 in human serum. The assay performance has been evaluated at 10 hospitals. Clinical specificity was evaluated by measuring 972 hospitalized patients and 586 donors of a normal population. Clinical sensitivity was assessed on 513 confirmed cases of SARS-CoV-2 by RT-PCR. Results: The assays demonstrated satisfied assay precision with coefficient of variation of less than 4.45%. Inactivation of specimen did not affect assay measurement. SARS-CoV-2 IgM showed clinical specificity of 97.33 and 99.49% for hospitalized patients and the normal population respectively, and SARS-CoV-2 IgG showed clinical specificity of 97.43 and 99.15% respectively. SARS-CoV-2 IgM showed clinical sensitivity of 82.54, 92.93, and 84.62% before 7 days, 7-14 days, and after 14 days respectively, since onset of symptoms, and SARS-CoV-2 IgG showed clinical sensitivity of 80.95, 97.98, and 99.15% respectively at the same time points above. Conclusions: We have developed fully automated immunoassays for detecting SARS-CoV-2 IgM and IgG antibodies in human serum. The assays demonstrated high clinical specificity and sensitivity, and add great value to nucleic acid testing in fighting against the global pandemic of the SARS-CoV-2 infection.