Journal: Analytical Biochemistry

Article Title: DNA aptamer selection for SARS-CoV-2 spike glycoprotein detection

doi: 10.1016/j.ab.2022.114633

Figure Lengend Snippet: FLAA specificity and detection parameters . A) Schematics of the FLAA procedure. Step 1: 5′-amino-C6-modified C7 aptamer was immobilized on the surface of maleic anhydride-activated multiwell plates as capture agent. Step 2: The purified recombinant SARS-CoV-2 S or negative binding control protein were added to the C7-containing plates. Step 3: Fluorescein-labeled C9 was added as detection agent. Step 4: After step 3 the multiwells are washed with TNa buffer. The multiwell plates are incubated with 7 M urea and volume is transferred to black plates. B) FLAA test based on the C7 and C9 aptamers. Purified recombinant S protein (250 nM) was added and incubated before addition of FAM-C9. Milk casein, human ACE2 and mouse IgG, were used as non-related controls to evaluate the FLAA specificity in 10-fold diluted human saliva. The graphs represent the mean and standard deviation from three independent experiments analyzed by one-way ANOVA with post hoc Tukey's multiple comparisons test (95% CI). Asterisks indicate statistical significance (n = 3, p < 0.0001). C) The FLAA test does not detect unrelated proteins and other common cold recombinant surface virus proteins. Milk casein, egg lysozyme, Human Respiratory Syncytial virus (RSV) glycoprotein G and Human Coronavirus (HCoV-NL63) S protein (250 nM) were added and incubated before addition of FAM-C9. The graphs represent the mean and standard deviation from three independent experiments analyzed by one-way ANOVA with post hoc Tukey's multiple comparisons test (95% CI). Asterisks indicate statistical significance (n = 3, p < 0.0001). D) FLAA signal is concentration-dependent of S protein. A FLAA test based was developed in 96-microwell plate format using aptamer C7 as capturing agent and FAM-labeled C9 as detection agent. . The FLAA concentration curve (0 nM–600 nM) of S protein (Black circles) showed a simple linear regression (R2 = 0.94). Background fluorescence from the well without S protein was subtracted from the measurements. BSA (600 nM) was used as negative control (Black triangle). Plotted data represents the mean and standard deviation of three independent experiments (n = 3).

Article Snippet: Briefly, 100 pmole of 5′-amino-C6-modified C7 aptamer was immobilized on Pierce™ maleic anhydride activated plates (Thermo Fisher Scientific) as capture agent.

Techniques: Modification, Purification, Recombinant, Binding Assay, Labeling, Incubation, Standard Deviation, Concentration Assay, Fluorescence, Negative Control