Journal: Genes & Diseases

Article Title: Dual-mode aptamer-driven biosensing platform for ultrasensitive and mutation-resilient detection of the SARS-CoV-2 nucleocapsid protein

doi: 10.1016/j.gendis.2025.101943

Figure Lengend Snippet: Workflow of NP14 aptamer screening and development of the MD ELAAA detection platform. (A) Schematic illustration of the X-aptamer protein SELEX process for isolating aptamers. (B) Schematic illustration of the ultrasensitive detection of the SARS-CoV-2 N protein via the MD ELAAA platform.

Article Snippet: X-Aptamer libraries were acquired from AM Biotechnologies (Houston, Texas, USA); His-Tag magnetic beads (Invitrogen, DynabeadsTM His-Tag Isolation & Pulldown, 10103D), SARS-CoV-2 N protein, and anti-SARS-CoV-2 N protein monoclonal antibodies (anti-SARS-CoV-2 N protein mAb, Cat: 40143-MM05, 40588-R001) were purchased from Sino Biological.

Techniques:

Journal: Genes & Diseases

Article Title: Dual-mode aptamer-driven biosensing platform for ultrasensitive and mutation-resilient detection of the SARS-CoV-2 nucleocapsid protein

doi: 10.1016/j.gendis.2025.101943

Figure Lengend Snippet: Binding affinity and stability characterization of the NP14 aptamer. (A) Magnetic bead (12.5 mg/mL, 3 μL) flow assay for the binding of the aptamer to the His-tag SARS-CoV-2 N protein (1 μg). (B) Flow cytometry analysis of the binding of 300 nM FAM-labeled aptamer NP14 to magnetic beads coated with the SARS-CoV-2 N protein. (C) Flow cytometry analysis of the binding of 300 nM FAM-labeled NP14 to magnetic beads coated with the SARS-CoV-2 N protein at different temperatures (4 °C, 25 °C, and 37 °C). (D) The binding affinity of NP14 for the SARS-CoV-2 N protein was validated via the use of 2 μg/mL SARS-CoV-2 N protein and biotin-labeled NP14 at different concentrations (0, 2.5, 5, 10, 20, 50, 100, 150, and 200 nM). (E) Determination of the Kd value of aptamer NP14 (15.625, 31.25, 62.5, 125, 250, and 500 nM) via surface plasmon resonance. (F) Confocal analysis of 300 nM FAM-labeled aptamer NP14 with SARS-CoV-2 N protein-coated magnetic beads (scale bar = 30 μm).

Article Snippet: X-Aptamer libraries were acquired from AM Biotechnologies (Houston, Texas, USA); His-Tag magnetic beads (Invitrogen, DynabeadsTM His-Tag Isolation & Pulldown, 10103D), SARS-CoV-2 N protein, and anti-SARS-CoV-2 N protein monoclonal antibodies (anti-SARS-CoV-2 N protein mAb, Cat: 40143-MM05, 40588-R001) were purchased from Sino Biological.

Techniques: Binding Assay, Flow Cytometry, Labeling, Magnetic Beads, SPR Assay

Journal: Genes & Diseases

Article Title: Dual-mode aptamer-driven biosensing platform for ultrasensitive and mutation-resilient detection of the SARS-CoV-2 nucleocapsid protein

doi: 10.1016/j.gendis.2025.101943

Figure Lengend Snippet: Structural basis and binding mechanism of NP14 interaction with the SARS-CoV-2 N protein. (A) Molecular simulation of the binding mode between aptamer NP14 and the SARS-CoV-2 N protein ( http://www.rcsb.org , ID:6VYO) via AutoDock. (B) Enlarged view of the presumed binding area. (C) Nucleic acid sequences and corresponding amino acids involved in the docking model. (D) Secondary structure simulation of aptamer NP14 via the Nupack web server at 37 °C. (E) Secondary structure simulation of the truncated chains NP14a via the Nupack web server at 37 °C. (F) Secondary structure simulation of the truncated chains NP14b via the Nupack web server at 37 °C. (G) Binding analysis of NP14 with truncated NP14a, NP14b, and base-mutated 400 nM NP14a1, NP14a2, NP14a3, NP14a4, NP14b1, NP14b2, NP14b3, NP14b4, and NP14b5 to the SARS-CoV-2 N protein by ELONA. Data were presented as mean ± standard deviation of triplicate results ( n = 3). The NP14 control: ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (H) Circular dichroism spectroscopy of AS1411 (20 μM) and NP14 (10 μM) was performed in PBS buffer (0.01 M, pH = 7.4) at wavelengths ranging from 220 to 320 nm. (I) Domain organization of the SARS-CoV-2 N protein, with numbers indicating domain boundaries. (J) Immunomagnetic beads (40 μL, 10 mg/mL) labeled with Flag antibodies against the truncated overexpressed protein were reacted with 300 nM biotin-labeled NP14 to assess binding. Data were presented as mean ± standard deviation of triplicate results ( n = 3). Compared with the blank control: ∗∗∗∗ p < 0.0001. (K) 250 nM biotin-labeled NP14 was mixed with 250 nM unlabeled N1, A58, A61 and competitive binding was analyzed by ELONA. Data were presented as mean ± standard deviation of four replicate results ( n = 4). Compared with the NP14: ns, not significant; ∗∗∗ p < 0.001. (L) Evaluation of the binding affinity for truncated proteins containing the NTD region at different concentrations of NP14 (0, 2, 5, 10, 20, 50, and 100 nM). Data were presented as mean ± standard deviation of triplicate results ( n = 3).

Article Snippet: X-Aptamer libraries were acquired from AM Biotechnologies (Houston, Texas, USA); His-Tag magnetic beads (Invitrogen, DynabeadsTM His-Tag Isolation & Pulldown, 10103D), SARS-CoV-2 N protein, and anti-SARS-CoV-2 N protein monoclonal antibodies (anti-SARS-CoV-2 N protein mAb, Cat: 40143-MM05, 40588-R001) were purchased from Sino Biological.

Techniques: Binding Assay, Standard Deviation, Control, Circular Dichroism, Spectroscopy, Labeling

Journal: Genes & Diseases

Article Title: Dual-mode aptamer-driven biosensing platform for ultrasensitive and mutation-resilient detection of the SARS-CoV-2 nucleocapsid protein

doi: 10.1016/j.gendis.2025.101943

Figure Lengend Snippet: Specificity and cross-variant recognition of NP14 for the SARS-CoV-2 N protein. (A) ELONA method detection mode diagram. (B) NP14 labeled with 400 nM biotin was used with various proteins (1 μg/mL): SARS-CoV N protein, human coronavirus (HCoV) 229E, OC43, HKU1, SARS-CoV-2 receptor-binding domain (RBD), alpha-fetoprotein (AFP), interleukin-4 (IL-4), bovine serum albumin (BSA), and influenza (InFlu) A and B proteins, to validate the specificity of NP14 via ELISA. Data were presented as mean ± standard deviation of triplicate results ( n = 3). Compared with the SARS-CoV-2 N protein: ns, not significant; ∗∗∗∗ p < 0.0001. (C) Direct detection of SARS-CoV-2 N protein binding activity at various concentrations (0, 0.5, 1, 5, 10, 20, 50, 100, 200, 500, 800, and 1000 ng/mL) via the ELONA platform. Data were presented as mean ± standard deviation of triplicate results ( n = 3). (D – L) Detection of NP14 (biotin-labeled, 400 nM) binding to N recombinant proteins from SARS-CoV-2 variants at different concentrations (0, 5, 10, 20, 50, 100, 200, 500, and 1000 ng/mL) on the direct ELONA platform. Variants included (D) alpha, (E) beta, (F) gamma, (G) delta, (H) omicron B.1.640, (I) omicron BA.2, (J) lambda, (K) omicron BA.1, and (L) omicron BA.4.

Article Snippet: X-Aptamer libraries were acquired from AM Biotechnologies (Houston, Texas, USA); His-Tag magnetic beads (Invitrogen, DynabeadsTM His-Tag Isolation & Pulldown, 10103D), SARS-CoV-2 N protein, and anti-SARS-CoV-2 N protein monoclonal antibodies (anti-SARS-CoV-2 N protein mAb, Cat: 40143-MM05, 40588-R001) were purchased from Sino Biological.

Techniques: Variant Assay, Labeling, Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Protein Binding, Activity Assay, Recombinant

Journal: Genes & Diseases

Article Title: Dual-mode aptamer-driven biosensing platform for ultrasensitive and mutation-resilient detection of the SARS-CoV-2 nucleocapsid protein

doi: 10.1016/j.gendis.2025.101943

Figure Lengend Snippet: Comparative sensitivity and specificity of antibody–antibody versus antibody–aptamer sandwich assays. (A) Standard curve for the sandwich assay (1 μg/mL antibody) using the SARS-CoV-2 N protein at various concentrations (0, 0.1, 0.5, 1, 5, 10, 20, 50, 100, 200, 500, and 1000 ng/mL). Data were presented as mean ± standard deviation of triplicate results ( n = 3). (B) Standard curve of the SARS-CoV-2 N protein in the antibody‒aptamer sandwich mode using SARS-CoV-2 N protein at various concentrations (0, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, 500, and 1000 ng/mL). Data were presented as mean ± standard deviation of triplicate results ( n = 3). (C) Specificity validation with multiple proteins (1 μg/mL), including: SARS-CoV-2 receptor-binding domain (RBD), alpha-fetoprotein (AFP), interleukin-4 (IL-4), bovine serum albumin (BSA), influenza (InFlu) A and B proteins, to validate the specificity of the antibody–antibody (1 μg/mL) sandwich assay. Data were presented as mean ± standard deviation of triplicate results ( n = 3). Compared with the blank control: ns, not significant; ∗∗ p < 0.01 and ∗∗∗∗ p < 0.0001. (D) Validation was performed using multiple proteins at a concentration of 1 μg/mL, including: SARS-CoV-2 RBD, AFP, IL-4, BSA, InFlu A and B proteins, to validate the specificity of the antibody (1 μg/mL)-aptamer (200 nM) sandwich assay. Data were presented as mean ± standard deviation of triplicate results ( n = 3). Compared with the blank control: ns, not significant; ∗∗∗∗ p < 0.0001.

Article Snippet: X-Aptamer libraries were acquired from AM Biotechnologies (Houston, Texas, USA); His-Tag magnetic beads (Invitrogen, DynabeadsTM His-Tag Isolation & Pulldown, 10103D), SARS-CoV-2 N protein, and anti-SARS-CoV-2 N protein monoclonal antibodies (anti-SARS-CoV-2 N protein mAb, Cat: 40143-MM05, 40588-R001) were purchased from Sino Biological.

Techniques: Standard Deviation, Biomarker Discovery, Binding Assay, Control, Concentration Assay

Journal: Genes & Diseases

Article Title: Dual-mode aptamer-driven biosensing platform for ultrasensitive and mutation-resilient detection of the SARS-CoV-2 nucleocapsid protein

doi: 10.1016/j.gendis.2025.101943

Figure Lengend Snippet: Analytical performance of the MD ELAAA platform in detecting the SARS-CoV-2 N protein and viral cultures. (A) Schematic illustration of the modulation of the Ag shell layer thickness in core–shell AuNFs@Ag nanostructures leading to changes in the localized surface plasmon resonance (LSPR) and light scattering intensity. (B) Standard curve of the MD ELAAA method for different SARS-CoV-2 N proteins (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.5, 1, 2, and 5 ng/mL). Data were presented as mean ± standard deviation of triplicate results ( n = 3). (C) Validation was performed using multiple proteins at a concentration of 1 ng/mL, including: SARS-CoV-2 receptor-binding domain (RBD), alpha-fetoprotein (AFP), interleukin-4 (IL-4), bovine serum albumin (BSA), influenza (InFlu) A and B proteins, to validate the specificity of the MD ELAAA platform. Data were presented as mean ± standard deviation of triplicate results ( n = 3). The blank control: ns, not significant; ∗∗∗∗ p < 0.0001. (D) Standard curve of the MD ELAAA method for SARS-CoV-2 virus cultures at different concentrations (0, 1, 2, 5, 10, 20, 50, 100, and 200 TCID 50 /mL). Data were presented as mean ± standard deviation of triplicate results ( n = 3). (E) Standard curve of the ELAAA method for SARS-CoV-2 virus cultures at different concentrations (0, 10, 20, 50, 100, 200, 300, 500, and 1000 TCID 50 /mL). Data were presented as mean ± standard deviation of triplicate results ( n = 3).

Article Snippet: X-Aptamer libraries were acquired from AM Biotechnologies (Houston, Texas, USA); His-Tag magnetic beads (Invitrogen, DynabeadsTM His-Tag Isolation & Pulldown, 10103D), SARS-CoV-2 N protein, and anti-SARS-CoV-2 N protein monoclonal antibodies (anti-SARS-CoV-2 N protein mAb, Cat: 40143-MM05, 40588-R001) were purchased from Sino Biological.

Techniques: SPR Assay, Standard Deviation, Biomarker Discovery, Concentration Assay, Binding Assay, Control, Virus

Journal: iScience

Article Title: Intranasal replicating adenovirus type 4-vectored SARS-CoV-2 vaccines induce durable and efficacious responses in preclinical testing

doi: 10.1016/j.isci.2026.115433

Figure Lengend Snippet: IN Ad4-SARS-CoV-2 induces binding antibodies on the nasal mucosa Heatmap depicting binding IgA (left), IgG (center) and IgM (right) responses observed in hamster nasal wash (top), BAL (center), and serum (bottom) for each immunization group (columns) against the indicated SARS-CoV antigens (rows), including Spike (S), the S2 and receptor-binding domain (RBD), and Nucleocapsid protein. Mean response values in each group of 6 animals are graphed.

Article Snippet: For surface staining cells were harvested using 0.01 M EDTA in PBS 48 h after infection and stained with mouse anti-SARS-CoV-2 spike monoclonal antibody (Sino Biological, Inc., Beijing, China) (50 μL at 1.0 μg/mL final concentration), washed twice with phosphate buffered saline (PBS) (Quality Biological, Gaithersburg, MD, USA) containing 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA), then stained with a goat anti-mouse IgG PE-conjugated secondary antibody (BioLegend, San Diego, CA, USA) (100 μL at 2.0 μg/mL final concentration) at 37°C for 30 min each.

Techniques: Binding Assay

Journal: iScience

Article Title: Intranasal replicating adenovirus type 4-vectored SARS-CoV-2 vaccines induce durable and efficacious responses in preclinical testing

doi: 10.1016/j.isci.2026.115433

Figure Lengend Snippet: IN Ad4-Spike immunization limits weight loss following SARS-CoV-2 challenge Average percent body weight relative to the day of challenge, at either 56 days or 174 days following the first immunization. Hamsters were weighed once individually per day. Dots represent median and bars represent standard error for each group ( N = 10 hamsters).

Article Snippet: For surface staining cells were harvested using 0.01 M EDTA in PBS 48 h after infection and stained with mouse anti-SARS-CoV-2 spike monoclonal antibody (Sino Biological, Inc., Beijing, China) (50 μL at 1.0 μg/mL final concentration), washed twice with phosphate buffered saline (PBS) (Quality Biological, Gaithersburg, MD, USA) containing 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA), then stained with a goat anti-mouse IgG PE-conjugated secondary antibody (BioLegend, San Diego, CA, USA) (100 μL at 2.0 μg/mL final concentration) at 37°C for 30 min each.

Techniques:

Journal: iScience

Article Title: Intranasal replicating adenovirus type 4-vectored SARS-CoV-2 vaccines induce durable and efficacious responses in preclinical testing

doi: 10.1016/j.isci.2026.115433

Figure Lengend Snippet: IN immunization with Ad4-Anc reduces lung pathology following SARS-CoV-2 challenge Hamsters from each group were sacrificed four days after challenge (day 60 or 178) to assess disease severity in the lungs. Scores ranging from 1 to 5 were assigned based on pneumonia severity and IHC staining in the bronchial epithelium and alveoli. Average scores for each group ( N = 4 hamsters), and the percent of the lung affected by pneumonia, are indicated.

Article Snippet: For surface staining cells were harvested using 0.01 M EDTA in PBS 48 h after infection and stained with mouse anti-SARS-CoV-2 spike monoclonal antibody (Sino Biological, Inc., Beijing, China) (50 μL at 1.0 μg/mL final concentration), washed twice with phosphate buffered saline (PBS) (Quality Biological, Gaithersburg, MD, USA) containing 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA), then stained with a goat anti-mouse IgG PE-conjugated secondary antibody (BioLegend, San Diego, CA, USA) (100 μL at 2.0 μg/mL final concentration) at 37°C for 30 min each.

Techniques: Immunohistochemistry

Journal: iScience

Article Title: Intranasal replicating adenovirus type 4-vectored SARS-CoV-2 vaccines induce durable and efficacious responses in preclinical testing

doi: 10.1016/j.isci.2026.115433

Figure Lengend Snippet: IN Ad4-Spike induces the durable restriction of SARS-CoV-2 replication after transmission. Weight loss and viral replication data following transmission at day 268 (A) Percent body weight relative to the day of challenge, represented as in . Dots represent the median for each group ( N = 6 hamsters) and bars represent standard error (B) AUC of viral copies measured in oropharyngeal swabs of individual hamsters, as represented in . Values for each hamster are indicated with dots, the median AUC value for each group ( N = 6 hamsters) is represented as a black line. The dotted line indicates the AUC limit of detection over 14 days (12,600 copies). (C) Curves representing viral replication over time after challenge. The limit of detection is depicted by the dotted line (10 copies per m L). Dots represent medians and bars represent standard error for each group ( N = 6 animals). The Wilcoxon rank-sum test was used for pairwise comparisons to generate the indicated p values.

Article Snippet: For surface staining cells were harvested using 0.01 M EDTA in PBS 48 h after infection and stained with mouse anti-SARS-CoV-2 spike monoclonal antibody (Sino Biological, Inc., Beijing, China) (50 μL at 1.0 μg/mL final concentration), washed twice with phosphate buffered saline (PBS) (Quality Biological, Gaithersburg, MD, USA) containing 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA), then stained with a goat anti-mouse IgG PE-conjugated secondary antibody (BioLegend, San Diego, CA, USA) (100 μL at 2.0 μg/mL final concentration) at 37°C for 30 min each.

Techniques: Transmission Assay