Journal: Analytical Chemistry

Article Title: Simple Nano-Luciferase-Based Assay for the Rapid and High-Throughput Detection of SARS-CoV-2 3C-Like Protease

doi: 10.1021/acs.analchem.2c02590

Figure Lengend Snippet: Principle of this assay for 3CLpro detection. (A) MBs-peptide-nLuc was added into PBS containing 3CLpro standards (or virus lysate). (B) Peptide on the surface of MBs-peptide-nLuc was cleaved by 3CLpro, and nLuc was released into the supernatant. (C) Supernatant was transferred into the 96-well plate pre-added with the luciferase substrate, and (D) the signal of each well was detected using the microplate reader.

Article Snippet: 3CLpro-His recombinant protein was purchased from Sino Biological Inc. HRP conjugated goat anti-mouse IgG (H+L), matrix metalloprotein 9 (MMP 9), and caspase 9 were purchased from Proteintech Group, Inc. Ultrasensitive ECL substrate was purchased from Thermo.

Techniques: Luciferase

Journal: Analytical Chemistry

Article Title: Simple Nano-Luciferase-Based Assay for the Rapid and High-Throughput Detection of SARS-CoV-2 3C-Like Protease

doi: 10.1021/acs.analchem.2c02590

Figure Lengend Snippet: Selection and optimization of the peptide substrate sequence of 3CLpro. (A) SARS-CoV-2 genome and 3CLpro potential restriction sites. (B) Bioinformation analysis revealed 11 potential cleavage sites of 3CLpro. (C) Prediction of the most likely amino acid sequence of the 3CLpro hydrolysis site.

Article Snippet: 3CLpro-His recombinant protein was purchased from Sino Biological Inc. HRP conjugated goat anti-mouse IgG (H+L), matrix metalloprotein 9 (MMP 9), and caspase 9 were purchased from Proteintech Group, Inc. Ultrasensitive ECL substrate was purchased from Thermo.

Techniques: Selection, Sequencing

Journal: Analytical Chemistry

Article Title: Simple Nano-Luciferase-Based Assay for the Rapid and High-Throughput Detection of SARS-CoV-2 3C-Like Protease

doi: 10.1021/acs.analchem.2c02590

Figure Lengend Snippet: Optimization of experimental parameters. (A) Feasibility of this assay for 3CLpro detection. The incubation time of GST-peptide-nLuc with 3CLpro was 7.5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. 3CLpro: 200 ng/mL. (B) Maximum binding quantity of GST-peptide-nLuc to the surface of 1 μg of GSH-MBs. (C) Effect of reaction buffer, and (D) the different incubation times of 3CLpro with GST-peptide-nLuc on signal intensity. 3CLpro: 200 ng/mL. The error bars represent the standard deviation of three independent measurements.

Article Snippet: 3CLpro-His recombinant protein was purchased from Sino Biological Inc. HRP conjugated goat anti-mouse IgG (H+L), matrix metalloprotein 9 (MMP 9), and caspase 9 were purchased from Proteintech Group, Inc. Ultrasensitive ECL substrate was purchased from Thermo.

Techniques: Incubation, Binding Assay, Standard Deviation

Journal: Analytical Chemistry

Article Title: Simple Nano-Luciferase-Based Assay for the Rapid and High-Throughput Detection of SARS-CoV-2 3C-Like Protease

doi: 10.1021/acs.analchem.2c02590

Figure Lengend Snippet: (A) Sensitivity of this assay for 3CLpro detection. Statistical analysis was performed using Student’s t -test. ** P ≤ 0.01. (B) Plots of signal vs the log values of different concentrations of 3CLpro. (C) Specificity of this assay for 3CLpro detection. 3CLpro: 20 ng/mL, MMP 9: 100 ng/mL, Caspase 9: 100 ng/mL, Trypsin: 10 μg/mL. (D) Inhibition efficiencies of different concentrations of Ebselen on the activity of 3CLpro. 3CLpro: 200 ng/mL. The error bars represent the standard deviation of three independent measurements.

Article Snippet: 3CLpro-His recombinant protein was purchased from Sino Biological Inc. HRP conjugated goat anti-mouse IgG (H+L), matrix metalloprotein 9 (MMP 9), and caspase 9 were purchased from Proteintech Group, Inc. Ultrasensitive ECL substrate was purchased from Thermo.

Techniques: Inhibition, Activity Assay, Standard Deviation