Rapid Detection of Spike Protein Receptor Binding Region of SARS-CoV-2 and Its Variants Using a Nanosheet Probe

Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 9, 2025

Strategies for the rapid detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are critically needed due to conventional methods' limitations: narrow range, virus mutation-induced failure, time-consuming, and complex operations. Herein, we propose a method utilizing CuInP2S6 nanosheet probes Bio-Layer Interferometry (BLI) technology (5–10 min), noninvasive, broad-spectrum SARS-CoV-2 spike receptor binding domain (RBD) in human saliva. The nanoprobe exhibits higher affinity RBD compared most saliva proteins, allowing it be immobilized on BLI sensors easier protein elution. An eluent buffer containing Tween-20 salts was employed separate salivary proteins while retaining probes. This system can detect across broad spectrum low limit 25 ng/mL (S/N = 3) less than 10 min. To validate this system, experiments with pseudoviruses showed accurate identification RBD. Molecular dynamics simulations elucidated mechanism selective In conclusion, conceptual study based an situ strategy inorganic nanoprobes alongside techniques convenient, its variants. is anticipated inspire design implementation pathogens future.

Language: Английский

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SARS-CoV-2 and Coronaviruses: Understanding Transmission, Impact, and Strategies for Prevention and Treatment

Drugs and Drug Candidates, Journal Year: 2025, Volume and Issue: 4(1), P. 5 - 5

Published: Feb. 10, 2025

COVID-19, first identified in December 2019 Wuhan, China, is caused by the SARS-CoV-2 virus, a pathogen that primarily targets respiratory system and can lead to severe conditions such as acute distress syndrome (ARDS). Among seven coronaviruses known infect humans, three—SARS-CoV, MERS-CoV, SARS-CoV-2—are associated with illness significant morbidity. an enveloped, single-stranded RNA virus utilizes angiotensin-converting enzyme 2 (ACE2) receptor for cellular entry. The genetic sequence of highly mutable, leading emergence variants alter disease pathology transmission dynamics. World Health Organization (WHO) has classified these mutations into concern (VOCs), interest (VOIs), under monitoring (VUMs). This review provides in-depth analysis both historical emerging variants, summarizes recent advancements diagnostic methods detection, discusses current therapeutic strategies particular focus on virus-like particle (VLP) vaccines developed years. Additionally, we highlight ongoing approaches their implications managing COVID-19.

Language: Английский

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Development and evaluation of MMIRA-CRISPR/Cas13a-MBQD assay for the detection of influenza A/B viruses and SARS-CoV-2

Future Virology, Journal Year: 2025, Volume and Issue: unknown, P. 1 - 9

Published: Feb. 24, 2025

Language: Английский

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Sensitive amperometric immunosensor for pathogen antigen based on MoS2@AuNPs assembling dual-peptide as bioprobes with significant dual signal amplification

Analytica Chimica Acta, Journal Year: 2025, Volume and Issue: 1355, P. 344015 - 344015

Published: April 6, 2025

Language: Английский

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Bioscreening specific peptide-expressing phage and its application in sensitive dual-mode immunoassay of SARS-CoV-2 spike antigen

Talanta, Journal Year: 2023, Volume and Issue: 266, P. 125093 - 125093

Published: Aug. 18, 2023

Language: Английский

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Advances in Surrogate Neutralization Tests for High-Throughput Screening and the Point-of-Care

Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: March 4, 2025

InfoMetricsFiguresRef.SI Analytical ChemistryASAPArticle This publication is Open Access under the license indicated. Learn More CiteCitationCitation and abstractCitation referencesMore citation options ShareShare onFacebookXWeChatLinkedInRedditEmailBlueskyJump toExpandCollapse ReviewMarch 4, 2025Advances in Surrogate Neutralization Tests for High-Throughput Screening Point-of-CareClick to copy article linkArticle link copied!Simon StreifSimon StreifInstitute of Chemistry, Chemo- Biosensors, University Regensburg, Universitaetsstrasse 31, 93053 GermanyMore by Simon StreifView Biographyhttps://orcid.org/0000-0001-6081-7571Antje J. Baeumner*Antje BaeumnerInstitute Germany*Email: [email protected]More Antje BaeumnerView Biographyhttps://orcid.org/0000-0001-7148-3423Open PDFAnalytical ChemistryCite this: Anal. Chem. 2025, XXXX, XXX, XXX-XXXClick citationCitation copied!https://pubs.acs.org/doi/10.1021/acs.analchem.5c00666https://doi.org/10.1021/acs.analchem.5c00666Published March 2025 Publication History Received 29 January 2025Accepted 20 February 2025Revised 10 2025Published online 4 2025review-article© The Authors. Published American Chemical Society. licensed CC-BY 4.0 . License Summary*You are free share (copy redistribute) this any medium or format adapt (remix, transform, build upon) material purpose, even commercially within parameters below:Creative Commons (CC): a Creative license.Attribution (BY): Credit must be given creator.View full license*DisclaimerThis summary highlights only some key features terms actual license. It not has no legal value. Carefully review before using these materials. underCC-BY share(copy adapt(remix, below: Attribution *DisclaimerThis creator. View ACS Publications© SocietySubjectswhat subjects Article automatically applied from Subject Taxonomy describe scientific concepts themes article. Assays Biopolymers Immunology Peptides proteins SARS-CoV-2 Special IssuePublished as part Chemistry special issue "Fundamental Applied Reviews 2025".IntroductionClick section linkSection copied!Serological testing long played crucial role disease management clinical diagnostics. Both infection with viruses vaccination mediate humoral immune response, including generation specific antibodies. presence antibodies can therefore used qualitatively detect recent past infections quantitatively determine status patient. Antibody profiles generated different vary show differences versus vaccination, latter often uses one antigen rather than whole virus. Furthermore, while vaccinations result long-term immunity, others require booster shots every few years, yearly. Hence, necessity benefits serological typically tailored respective diseases.Diseases preventable through include, e.g., hepatitis A, influenza, SARS, chickenpox, measles, mumps, rubella, tetanus, poliomyelitis. Low mutation rates due constraints, such limited host range case (1) ensuring immunity memory B cell T persistence. Vaccination lead up 30 years longer (2) influenza viruses, especially rapid antigenic drift, necessitating extensive modeling predict most likely strains targeted annual vaccine. (3) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also shows high rates, facilitating its escape. COVID-19 pandemic caused virus sparked advancement innovation field regard both binding neutralizing antibody detection. consists four proteins: nucleocapsid (N), envelope (E), membrane (M), spike (S). Interaction mediated S protein receptor domain (RBD) S1 subunit binds human angiotensin converting enzyme (ACE2) receptor. (4) Cellular transmembrane protease serine (TMPRSS2) lysosomal cathepsin proteases cleave S2 subunits, followed fusion initiated subunit. (5) While target protein, majority directed against more specifically RBD. (6,7) Most vaccines make use observation introducing mRNA viral vectors induce expression (8,9) directly protein. (10) Testing antinucleocapsid thus check infections, unless an inactivated vaccine was used, all proteins. (11) Antispike anti-RBD antibodies, which produced after provide means assert might serve correlate protection (CoP). (12−14) For information on definition CoP other reviews Perry et al., (14) Sobhani (15) Plotkin (16) recommended.Two categories quantified, (nAbs). former includes certain antigen, that prevent infection, i.e., blocking virus–host interaction preventing host–cell fusion. tests mimic quantify indirectly via their ability block interaction. gold standard plaque reduction neutralization test (PRNT), live incubated patient serum dilutions prior addition cells expressing Infection results formation plaques, quantified manual automatic counting. PRNT50 value correlates dilution required reduce observed without 50%. makes PRNT conventional (cVNTs), micro (microNT), highly accurate but requires biosafety level (BSL) facility turn-around times 3 days. (17) In mentioned above, BSL-3 would most. To safety requirements at least BSL-2, pseudovirus-neutralization (pVNTs) have been developed, relying lentiviruses vesicular stomatitis pseudotyped responsible (18−20) pVNT development, Rocha (21) Sun (22) Vaidya (23) recommended. SARS-CoV-2, RBD identified main providing opportunity further simplify tests. (sVNTs), focus review, rely competitive relevant ACE2 RBD, respectively. These cell-free assays divided into two categories: high-throughput screening (HTS) point-of-care (POC) assays. They do facilities times, making widely available. COVID showed monitor development titers studies, insights SARS-CoV-2. Still, sVNTs endorsed regulatory agencies status, yet. (24) date, three granted emergency authorization (EUA) Food Drug Administration (FDA). (25) Lacking standardization validation during well difficulties defining titer currently hinders progress take advantage sVNTs. However, they technological answer broad needed situations. following, overview last provided. Advantages disadvantages formats critically analyzed, future potential, applicability toward discussed.Binding TestsClick copied!Quantification assessment minutes hours POC. Such secondary recognize sections molecules. Thus, approaches need account isotypes seroconversion. Specifically, immunoglobulin M (IgM) levels rise quickly drop shortly recovery, IgA IgG increase decrease reliable indicators reinfection. (26,27) Time-resolved combinations IgM detailed about seroconversion vaccination. selected specialized facilities. important note strength lies large parts population. because infectiousness precedes multiple days, unsuitable diagnostic stop spread disease. research improving sensitivity specificity, maintaining ease-of-use lateral flow assay (LFA), intensified over 5 remarkable novelties. example, Hossain al. (28) alkaline phosphatase (AP)-conjugated enable off-the-shelf glucometer strips incorporation maltose phosphatase. Streptavidin magnetic nanoparticles were modified biotinylated Alkaline yellow (pNPP) enzymatically degraded PO43–, phosphorylase stoichiometrically converts glucose, then amperometrically minipotentiostat. A similar approach investigated Peng (29) who AP-conjugated electrochemical detection commercial hand-held potentiostat. Their platform (SPEEDS) consisted streptavidin-coated carbon working electrode counter electrode, Ag/AgCl reference electrode. determined conversion p-aminophenyl phosphate p-aminophenol AP, oxidized p-quinonimine chronoamperometry. Other researchers focused new materials sensor Nunez (30) based zinc oxide nanorods min. positive charge ZnO it interesting option adsorption negatively charged Further optimization technology market ready, system stable 15 days so far. Nanorods involved Shen al.'s work, developed magnetofluid-integrated multicolor immunochip (MMI-chip). (31) eight liquid storage wells MMI-chip connected mineral oil layer reaction washing steps (Figure 1). horseradish peroxidase (HRP)-labeled oxidize substrate 3,3′,5,5′-tetramethylbenzidine (TMB), etches nanorods, decreasing length thereby changing absorption spectra, enabling semiquantitative visual read-out. Although being multistep assay, chip design sealed environment, minimizing external exposure increasing user-friendliness. Chip fabrication many publications. Kim microfluidic fluorescent LFA integrated dry reagents, mixer, vacuum pump. (32) sample first passes DyLight 550-labeled reagent chamber, mixed herringbone spike-labeled polystyrene particle chamber. particles captured zone pillars, fluorescence measurements inverted microscope.Figure 1Figure 1. Schematic illustration MMI-chip. (A) Structural diagram loading (I), solution (II III), (IV), (V–VII), signal (VII). (B) Magnetic bead transportation between mixing magnet. (C) Workflow peripheral blood. Reproduced permission ref (31). Copyright 2022 Society.High Resolution ImageDownload MS PowerPoint SlideMultiplexing offers another intriguing avenue, allowing simultaneous variety antigens, opening possibility screen several variants prevalent simultaneously. growing interest, continuously mutates dominating variant changes quickly. Several multiplexing platforms machine learning, barcode beads, nanopore sensing, more. (33−37) paper-based vertical (xVFA) Eryilmaz able five antigens <20 (33) device 3D-printed mobile-phone-based optical reader. neural network 89.5% accuracy 31 samples tested training. Importantly, addressed limitations choice panel later application. Serum panels consisting local sera small numbers may biased less variations status. Nan naked-eye readable microarray (NRM) thickness sensing nanoplasmonic ruler provided POC friendly multiplex 2). (35) NRM chips 16 (∼2 μL each) simultaneously <30 nanoparticle (AuNP) monolayer, where increased decreased reflectance, which, turn, measurable gray analysis smartphone images. manufacturing tape-based transfer AuNP deemed impractical, however. Exploiting effects, Huang immunosensor nanoporous hollow anti-IgG coated chip. (38) generate min amplification washing. Similarly, dual-affinity ratiometric quenching (DARQ) fast homogeneous format. Kilgour fluorescein-labeled rhodamine-labeled L, obtaining signals just (39) Liang combined Raman spectroscopy. (40) synthesized silver ultrathin shells embedded 4-mercaptobenzoic acid conjugated them HS-PEG-COOH EDC/NHS chemistry. enabled dual-mode qualitative quantitative SERS read-out portable spectrometer 785 nm laser strip. changed allow instead (41) excellent example demonstrating noncompetitive format, technologies universally applicable. fact, discussed next chapters evolved out found written Lee (42) Yari (43) Dong (44)Figure 2Figure 2. Visualization micromosaic (a) photograph illustrating ready-to-use (top) channels incubation (bottom). (b) representation immobilization layout (top). Smartphone image chip, (c) Heatmap box plots (bottom) showing random donors reopening China December (Cohort (35). 2023 SlideCommercial Binding Neutralizing copied!The diseases established, starting enzyme-linked immunosorbent (ELISA) (45−47) and, recently, LFAs. (48) pandemic, companies adapted existing tools Many resulting products gained EUA FDA, indicated distribution, go complete FDA process. Now, beginning outbreak, EUAs revoked, (49) off market. Once discontinued, finding principles behind difficult, datasheets become unavailable. cases, publications Web sites listing best obtain information. At point writing, still lists 75 EUA, issued April 2020 July 2024 traditional marketing authorization. (50) Of these, IgG, IgM, total levels. Only targeting antibodies: SCoV-2 Detect Ab ELISA (InBios International, Inc.), Diazyme CLIA Kit (Diazyme Laboratories, cPass Detection (GenScript). list reveals that, besides classification tests, there major groups tests: ELISAs chemiluminescence immunoassays (CLIAs) LFAs POCT. mainly microtiter plate microarrays labeled HRP, fluorescence, markers. predominant sVNT literature GenScript's Kit, RBD-conjugated HRP ACE2-coated 96-well microplate 3a). (51−54) Two separate 37 °C TMB, overall time ∼1 h. market, varying times; examples Leinco ImmunoRank (Leinco Technologies, Inc.) (53) TECO SARS-CoV-2-AK Neutralisation Test (TECOmedical). (51,53) Fluorescence read-outs shorten fully automated systems. Examples Roche Elecsys Anti-SARS-CoV-2 S, (55) ruthenium–RBD conjugates, streptavidin-conjugated detection, RBD-modified microbeads ACE2-ABEI. 18 34 min, Graninger compared seven robust correlation live-virus test, ACE2-RBD DiaPro displayed highest nAb (51) AuNPs markers reached Healgen Scientific Rapid Cassette (55,56) comprises AuNPs, line absence reflectance spectrum analyzer response. VERI-Q (MiCo BioMed) (57) captures AuNP-RBD bound ACE2-mouse-Fc goat antimouse line, requiring serum. sensitive obtained conjugates ichroma test. (58) capturing ACE2–biotin conjugate streptavidin susceptible interference biotin. Users advised 24 h stopping intake biotin supplements ensure functionality, concentrations 500 ng/mL shown interfere assay. When 1:5 factored in, slightly below threshold 3510 (14367 nM) Clinical Laboratory Standards Institute (CLSI) guideline (EP37), physiological concentration measured dose uptake. (59) Actually, common problem streptavidin–biotin interaction, should kept mind development. pursued PremaLabs Diagnostics NAb kit. According McLean capture neutralized fluorescently (60) ACE2, site antibody. appears problematic, position could theory causing false negative result. An works blood, plasma. LFIA Finecare 2019-nCoV S-RBD advertised short (61)Figure 3Figure 3. Illustration principle kit Ahn biotin-based sVNT. Reprinted (62). Elsevier.High SlideTo compete available products, newly factor characteristics. (I) type, HTS consideration steps, time, equipment, interconnected cost (II) type serum, plasma, preferably (finger prick) blood (III) Desired sensitivity, qualitative, semiquantitative, Besides signaling agent, volume affects sensitivity. (IV) conjugation strategy needs adaptation viruses. intertwined purification strategy.New Concepts sVNTsClick following will discuss advantages sVNTs, taking possible product. better overview, grouped formats, subchapters concerning method cases signal-on sVNTs.High-Throughput copied! HRP-Based DetectionMany HRP-based published, conjugates. Correlations either complicating comparison formats. Table 1 conjugate, substrate, correlations easier comparison. immobilized His6-tag improved 3b) GenS

Language: Английский

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Development of an ELISA Test with High Diagnostic Accuracy for SARS-COV-2 Using Recombinant Nucleocapsid Protein Expressed in E. coli

Molecular Biotechnology, Journal Year: 2025, Volume and Issue: unknown

Published: April 7, 2025

Language: Английский

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Development of triplex loop-mediated isothermal amplification with immunochromatographic assay for rapid simultaneous detection three respiratory viruses

Microchemical Journal, Journal Year: 2025, Volume and Issue: unknown, P. 113763 - 113763

Published: April 1, 2025

Language: Английский

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Iso‐E‐Codelock: A Rebuilding‐free Electrochemical Chip with a Customizable Decoding Probe for Real‐Time and Portable Pathogen Diagnostics

Angewandte Chemie International Edition, Journal Year: 2024, Volume and Issue: 63(19)

Published: March 18, 2024

Abstract In order to realize portable pathogen diagnostics with easier quantitation, digitization and integration, we develop a ready‐to‐use electrochemical sensing strategy (Iso‐E‐Codelock) for real‐time detection of isothermal nucleic acid amplification. Bridged by branched DNA as codelock, the amplicon is transduced into increased current an probe, holding multiple advantages high sensitivity, selectivity, signal‐on response, “zero” background one‐pot operation. Through self‐designed instrument (BioAlex PHE−T), can be implemented on multichannel microchip output amplification curves just like expensive commercial PCR machine. The rebuilding‐free disposable component. branch codelock probe customized different targets designs. Such performance flexibility have been demonstrated utilizing four virus (SARS‐CoV‐2, African swine fever, FluA FluB) genes targets, two (3‐way 4‐way) DNAs probes.

Language: Английский

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Design and Development of an Antigen Test for SARS-CoV-2 Nucleocapsid Protein to Validate the Viral Quality Assurance Panels

Viruses, Journal Year: 2024, Volume and Issue: 16(5), P. 662 - 662

Published: April 24, 2024

The continuing mutability of the SARS-CoV-2 virus can result in failures diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for calibration and quality assurance inactivated variant panels provided to Radical Acceleration Diagnostics programs (RADx)-radical program awardees. A heuristic genetic analysis based on variant-defining mutations demonstrated lowest variance Nucleocapsid protein (Np)-C-terminal domain (CTD) across all variants. We then employed Shannon entropy method (Np) sequences collected from major variants, verifying CTD with lower (less prone mutations) than other Np regions. Polyclonal monoclonal antibodies were raised against this target antigen used develop an Enzyme-linked immunoassay (ELISA) test SARS-CoV-2. Blinded Viral Quality Assurance (VQA) comprised UV-inactivated variants (XBB.1.5, BF.7, BA.1, B.1.617.2, WA1) distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, B, IAV H1N1, IBV) assembled by RADx-rad core tested using ELISA described here. assay positive high sensitivity (limit detection: 1.72–8.78 ng/mL) negative panel. Epitope mapping identified 20 amino acid antigenic peptide Np-CTD that in-silico also predicted highest antigenicity. This work provides template bioinformatics select regions low propensity mutation (low entropy) robust ‘pan-variant’ antigen-based assays mutational rates.

Language: Английский

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