Journal: Biosensors

Article Title: Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

doi: 10.3390/bios12110978

Figure Lengend Snippet: Overall schematic of multiplex nucleic acid diagnostic tests for combating SARS-CoV-2 variants and other respiratory viruses.

Article Snippet: WarmStart SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (New England Biolabs, Inc., Ipswich, MA, USA) incorporates RNA purification of either nasal swabs or saliva into the kit’s workflow, targeting both the N and E genomic regions, with a color change from pink to yellow if amplification occurs.

Techniques: Multiplex Assay, Diagnostic Assay

Journal: Biosensors

Article Title: Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

doi: 10.3390/bios12110978

Figure Lengend Snippet: PCR techniques and applications. ( a ) Basic schematic of the core PCR amplification steps. The dsDNA containing the targets of interest are denatured into two separate ssDNA strands. Hybridization of primers complementary to target sequences of interest signals DNA polymerase to amplify ssDNA targets through thermocycling. ( b ) Adaptation of a standard laboratory centrifuge into a fully customizable disc that allows for PCR into 64 independent direct RT-qPCR across four independent loading units. Adapted with permission from Ref. . Copyright 2020, Royal Society of Chemistry. ( c ) The utilization of fluorescent microspheres (MPA enhanced ARMS-PCR platform) for the amplification of fluorophores on primers targeting genes of interest in the detection of SARS-CoV-2. Adapted with permission from Ref. . Copyright 2022, Elsevier. ( d ) Decreasing the signal-to-noise ratio of genomic target to background interference using the ddPCR platform. As shown in this platform, a series of microfluidic channels create individual droplets, which can then be separated into individual bioreactor units on a silicon chip. Adapted with permission from Ref. . Copyright 2021, Elsevier.

Article Snippet: WarmStart SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (New England Biolabs, Inc., Ipswich, MA, USA) incorporates RNA purification of either nasal swabs or saliva into the kit’s workflow, targeting both the N and E genomic regions, with a color change from pink to yellow if amplification occurs.

Techniques: Amplification, Hybridization, Quantitative RT-PCR

Journal: Biosensors

Article Title: Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

doi: 10.3390/bios12110978

Figure Lengend Snippet: CRISPR/Cas technologies and multiplexing applications. ( a ) Demonstration of cis-cleavage and trans-cleavage of CRISPR/Cas mechanism, as crRNA-M recognizes the mutant DNA (MT-DNA). Adapted with permission from Ref. . Copyright 2022, Elsevier. ( b ) Multiplex colorimetric assay triggered by LAMP (ON signal) and eliminating false positives by using a CRISPR/Cas9 system (OFF signal) to detect VOCs. Adapted with permission from Ref. . Copyright 2022, American Chemical Society. ( c ) The combination of nested RPA and CRISPR/Cas12a in the same reaction pot. Adapted with permission from Ref. . Copyright 2022, Wiley-VCH. ( d ) Multiple crRNAs targeting different regions of SARS-CoV-2 RNA with single reporter RNA type to improve the sensitivity of the assay. Adapted with permission from Ref. . Copyright 2021, Elsevier. ( e ) The magnetic beads conjugated with CRISPR/Cas complex triggered by target RNA and pulled down by the magnetic force into the microchamber, concentrating the reactions. Adapted with permission from Ref. . Copyright 2022, Springer Nature. ( f ) RT-PCR CRISPR-based diagnostic and microfluidic panel, mCARMEN, for parallelizing nucleic acid detection, utilizing a commercial chip Fluidigm, which can identify up to 21 viruses. Adapted with permission from Ref. . Copyright 2022, Springer Nature.

Article Snippet: WarmStart SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (New England Biolabs, Inc., Ipswich, MA, USA) incorporates RNA purification of either nasal swabs or saliva into the kit’s workflow, targeting both the N and E genomic regions, with a color change from pink to yellow if amplification occurs.

Techniques: CRISPR, Multiplexing, Mutagenesis, Multiplex Assay, Colorimetric Assay, Magnetic Beads, Reverse Transcription Polymerase Chain Reaction, Diagnostic Assay

Journal: Biosensors

Article Title: Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

doi: 10.3390/bios12110978

Figure Lengend Snippet: Fluorescence readout and applications. ( a ) Illustration of how fluorescent signals are generated to detect SARS-CoV-2 nucleic acids of interest. Fluorescent optical detection involves a fluorophore emitting a signal at a particular wavelength from an initiating, excitation wavelength. ( b ) Intercalation of dyes sandwiched between the hydrogen bonds of complementary nucleic acid sequences. ( c ) Analysis of melting curve profiles to detect and distinguish two targets in a multiplex assay utilizing intercalating dyes as a fluorescent signal. Adapted with permission from Ref. . Copyright 2021, the Authors. ( d ) FRET-based fluorescent detection has two distinct mechanisms: increasing the distance between a fluorophore and the quencher or decreasing the distance between two fluorophores to allow for energy transfer from one fluorophore to another. ( e ) A MoS 2 nanosheet-modified DMA platform designed to detect SARS-CoV-2 genomic material on the nM scale using a FRET-based optical detection method. This multiplex test enabled the detection of SARS-CoV-2 genetic material alongside HIV. Adapted with permission from Ref. . Copyright 2020, American Chemical Society. ( f ) A one-pot OPIPE assay utilizing pre-coated tubes for the detection of RdRp and E genes. The TaqMan probes used have different fluorophores that enable fluorescent detection after cleavage during the RT-PCR amplification process. Adapted with permission from Ref. . Copyright 2021, Elsevier. ( g ) Utilization of the Proofman enzyme, a protein that recognizes SNVs, which can cleave fluorophores away from restrictive quenchers on a primer specifically targeting mutations. This set-up is performed in a singular pot for multiple targets of interest. Adapted with permission from Ref. . Copyright 2021, Elsevier.

Article Snippet: WarmStart SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (New England Biolabs, Inc., Ipswich, MA, USA) incorporates RNA purification of either nasal swabs or saliva into the kit’s workflow, targeting both the N and E genomic regions, with a color change from pink to yellow if amplification occurs.

Techniques: Fluorescence, Generated, Multiplex Assay, Modification, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: Biosensors

Article Title: Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19

doi: 10.3390/bios12110978

Figure Lengend Snippet: Point-of-care technologies. ( a ) POCT for improving diagnostic testing accessibility to medical deserts, especially in lower income communities around the world. The use of smartphone technology, microfluidic integration, and/or continued LFA development is pertinent to empowering individuals without high-resource clinicians and hospitals nearby. ( b ) Smartphone-based detection for amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a. Adapted with permission from Ref. . Copyright 2021, Elsevier. ( c ) Smartphone-based detection of VOCs by PAM-targeting mutations. Adapted with permission from Ref. . ( d ) Lateral flow assay using nanoparticles. Adapted with permission from Ref. . Copyright 2020, Elsevier. ( e ) Lateral flow assay with RPA for low resource setting. Adapted with permission from Ref. . Copyright 2021, Elsevier. ( f ) 3D-printed integrated microfluidic chip for colorimetric detection. Adapted with permission from Ref. . Copyright 2021, Elsevier. ( g ) Microfluidic chip with LAMP-based portable assay. Adapted with permission from Ref. . Copyright 2020, the Authors.

Article Snippet: WarmStart SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (New England Biolabs, Inc., Ipswich, MA, USA) incorporates RNA purification of either nasal swabs or saliva into the kit’s workflow, targeting both the N and E genomic regions, with a color change from pink to yellow if amplification occurs.

Techniques: Diagnostic Assay, Amplification, CRISPR, Lateral Flow Assay

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The inhibition of the interaction between the spike protein S1 receptor-binding domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) receptor by kuwanon C (KC). Spike protein coated on a 96-well plate interacted with a preincubated mixture of the ACE2 receptor and ( A ) anti-SARS-CoV-2 spike S1 antibody as the positive control and ( B ) 0, 3.125, 6.25, 12.5, 25, 50, or 100 μM KC. The inhibition of the spike S1 RBD:ACE2 receptor interaction by KC was determined based on chemiluminescence measurements.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Inhibition, Binding Assay, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The global kinetic analysis of KC binding to biotinylated ( A ) spike S1 RBD- and ( B ) ACE2 receptor-immobilized BLI sensors. The kinetics for the binding of KC to the spike S1 RBD or the ACE2 receptor were measured by the association of 0, 50, 200, and 400 μM of KC in PBS containing 1% DMSO with immobilized spike S1 or ACE2 receptor and the subsequent dissociation in PBS containing 1% DMSO.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: The binding kinetics of KC to spike S1 RBD and ACE2 receptor.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: In silico docking simulation between KC and the spike protein/ACE2 receptor. KC was docked onto the SARS-CoV-2 spike protein and ACE2 receptor (PDB code: 6M0J) using AutoDock Vina. The pharmacophore of KC with each target proteins was analyzed using BIOVIA Discovery Studio Visualizer.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: In Silico

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: KC inhibits SARS-CoV-2 lentiviral pseudovirus infection in HEK293T cells stably expressing human ACE2 and TMPRSS2. ( A ) The cytotoxic effect of KC in HEK293T cells stably expressing human ACE2 and TMPRSS2 was determined using the MTT assay. HEK293T cells were cultured in 96-well plates (5 × 10 4 cells/well) for 18 h. ( B ) The ACE2 expression level in HEK293T cells was monitored during KC treatment using real-time quantitative PCR analysis. Then, WT or mutant (D614G) SARS-CoV-2 spike pseudovirus (at a final concentration of 1 × 10 4 TU/mL to each well) were mixed with different concentrations of KC (2 and 20 μM) or anti-SARS-CoV-2 antibody, and the mixtures were incubated at 37 °C for 1 h. Then, these mixtures were added to HEK293T cells. ( C , D ) Green fluorescent protein (GFP) expression levels using flow cytometry were assessed at 72 h after viral infection, scale bar = 100 μm. ( E , F ) The inhibitory effect of SARS-CoV-2 spike pseudovirus infection was determined by measuring GFP expression using flow cytometry and measured under a fluorescence microscope. Bar graph (mean ± SEM) statistics were determined from three experimental data sets using one-way ANOVA with Tukey’s post hoc test, *** p < 0.001, compared with the CON (KC-untreated) samples. ### p < 0.001, compared with the cell-only sample.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Infection, Stable Transfection, Expressing, MTT Assay, Cell Culture, Real-time Polymerase Chain Reaction, Mutagenesis, Concentration Assay, Incubation, Flow Cytometry, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: KC suppresses the infection of a clinical isolate of SARS-CoV-2 alpha strain (βCoV/Korea/KCDC03/2020) in Vero cells. Vero cells were cultured on 384-well plates (1.2 × 10 4 cells/well) for 24 h. Then, Vero cells were infected with SARS-CoV-2 (MOI 0.0125) immediately after being treated with serially diluted KC and incubated at 37 °C for 24 h. The cells were then stained using anti-SARS-CoV-2 nucleocapsid (N) primary antibody, Alexa Fluor 488-conjugated goat antirabbit IgG secondary antibody, and Hoechst 33342.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques: Infection, Cell Culture, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction

doi: 10.3390/ijms232012516

Figure Lengend Snippet: Schematic of the blockade of the SARS-CoV-2 spike S1 RBD:ACE2 receptor interaction by KC.

Article Snippet: The SARS-CoV-2 spike/ACE2 inhibitor screening assay kit, biotin-labeled recombinant protein ACE2 receptor, and spike protein S1 RBD (BPS Bioscience, San Diego, CA, USA) were purchased for the competitive ELISA assay and BLItz analysis.

Techniques:

Journal: iScience

Article Title: A micro-electroporation/electrophoresis-based vaccine screening system reveals the impact of vaccination orders on cross-protective immunity

doi: 10.1016/j.isci.2023.108086

Figure Lengend Snippet:

Article Snippet: WT SARS-CoV-2 spike , Sino Biological , Cat #40589-V08B1.

Techniques: Virus, Recombinant, Cream, Enzyme-linked Immunosorbent Assay, Luciferase, Lysis, Staining, Membrane, Western Blot, Software

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Kae prevents the invasion of SARS-CoV-2 particles into host cells. (A and B), immunofluorescence staining (A) or enzyme-linked immunosorbent assay (ELISA) analysis (B) of SARS-CoV-2 virus-like particles (VLPs) invasion into human induced pluripotent stem cells (hiPSC)-derived alveolar epithelial cells type II (AECII). Control: dimethyl sulfoxide (DMSO)-treated cells; spike (S) protein neutralizing antibody (S NAb): positive control; and Kae: 10 µM in DMSO.(C and D), the entry of SARS-CoV-2 VLPs into lungs of hACE2 transgenic mice evaluated by immunofluorescence staining (C) or luciferase activity (D).Control: 0.5% carboxymethylcellulose sodium-treated mice; ursodeoxycholic acid (UDCA): positive control; and Kae: indicated dosages in 0.5% carboxymethylcellulose sodium. (A and C), VLPs, ACE2, F-actin and nucleus are shown as indicated colors. Scale bar = 25 µm. (B), ***, p < 0.005 vs Blank. #, p < 0.05 and ##, p < 0.01 vs Control. (D), n = 4, ***, p < 0.005 vs Blank. ##, p < 0.01; and ###, p < 0.005 vs Control. ns: no statistical differences vs Control.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Virus, Derivative Assay, Control, Positive Control, Transgenic Assay, Luciferase, Activity Assay

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Kae targets coronaviral fusion. (A), flow cytometry analyses of the binding affinity between spike (S) protein receptor-binding domain (S-RBD) and HEK-293F cells expressing angiotensin-converting enzyme 2 (ACE2) in the presence or absence of Kae with the S protein neutralizing antibody (S NAb) as a positive control. ***, p < 0.005 vs Blank. ###, p < 0.005, and ns, no statistical differences vs Control. (B), impacts of Kae on SARS-CoV-2 endocytosis into Vero E6 cells with S NAb as a positive control. ***, p < 0.005, and ns, no statistical differences vs Control. (C, D and E), effects of Kae on membrane fusion mediated by SARS-CoV-2 variants (C), or SARS-CoV (D), or MERS-CoV (E) with transmembrane serine protease 2 (TMPRSS2) inhibitor, camostat mesylate (CM), as a positive control. Scale bar = 300 µm. **, p < 0.01; and ***, p < 0.005 vs Control. Control: dimethyl sulfoxide (DMSO)-treated cells; DPP4: dipeptidyl peptidase 4; ns: no statistical differences vs Control.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Flow Cytometry, Binding Assay, Expressing, Positive Control, Control, Membrane

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Kae interferes with the S2 subunit of SARS-CoV-2 spike protein. (A), immunoblots representing the cleavage and abundance of SARS-CoV-2 spike (S) protein induced by Kae, with camostat mesylate (CM) as a positive control. (B), thermal stability determinations for full-length S protein, S1 and S2 subunits of SARS-CoV-2 in the presence or absence of Kae. Control: dimethyl sulfoxide (DMSO)-treated cells.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Western Blot, Positive Control, Control

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Kae impairs HR1 and HR2 of SARS-CoV-2 S2 subunit. (A and B), circular dichroism (CD) spectra analyses of synthesized heptad repeat 1 (HR1) and heptad repeat 2 (HR2) peptide, the formation of six-helix bundle (6-HB, HR1+HR2), and impacts of Kae on 6-HB, HR1 and HR2 using isopropanol as solvent controls. (C, D, E and F), native polyacrylamide gel electrophoresis (N-PAGE) assessment of effects of Kae on HR1, HR2 and 6-HB. *, p < 0.05; **, p < 0.01; and ***, p < 0.005 vs Control. ns: no statistical differences vs Blank. (E and F), the stability of wildtype (WT) and mutant (MT) HR2 in the absence (E) or presence (F) of Kae compared to corresponding wildtype peptide. (G), atomic force microscopy (AFM) topographic images before and after adding Kae to HR1, HR2 and 6-HB. Scale bar = 4 µm. (C, D, F and G), Control: dimethyl sulfoxide (DMSO)-treated peptides.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Circular Dichroism, Synthesized, Solvent, Polyacrylamide Gel Electrophoresis, Control, Mutagenesis, Microscopy

Journal: Phytomedicine

Article Title: Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

doi: 10.1016/j.phymed.2023.154942

Figure Lengend Snippet: Schematic diagram of the inhibitory mechanism of Kae against SARS-CoV-2 invasion. Instead of blocking the interaction between angiotensin-converting enzyme 2 (ACE2) and spike (S) protein receptor-binding domain (S-RBD), Kae disrupts heptad repeat 1 (HR1) and heptad repeat 2 (HR2) segments in the S2 subunit of SARS-CoV-2 S protein, leading to inhibitions of viral fusion-mediated invasion of SARS-CoV-2.

Article Snippet: The procedure involved incubating 100 ng/ml of human Fc-tagged SARS-CoV-2 S-RBD protein (40592-V02H, Sino Biological) with either 2‰ DMSO or 10 µM Kae for 30 min, then adding the mixture to HEK-293F-ACE2 cells and incubating for an additional 30 min at 37˚C.

Techniques: Blocking Assay, Binding Assay