Journal: ACS Synthetic Biology

Article Title: Nanobody-Based Lateral Flow Assay for Rapid Zika Virus Detection

doi: 10.1021/acssynbio.4c00819

Figure Lengend Snippet: Scheme of nanobody-based sandwich lateral flow. When the ZIKV NS1 protein antigen is added, it initially forms a complex with the AuNP-conjugated ZIKV_NbD6. This antigen-nanobody-AuNP complex is then immobilized on the test line by the capture nanobody ZIKV_Nb32. Any remaining nanobody-AuNP complexes are subsequently immobilized on the control line by an anti-VHH antibody. The LFA is considered positive when both the test and control lines are visible to the naked eye. It is negative when only the control line is visible. The assay is invalid if the control line is not visible.

Article Snippet: The ZIKV SPH2015 NS1 target protein was purchased from Sinobiological (cat: 40544-V07H, Figure S7 ).

Techniques: Control

Journal: ACS Synthetic Biology

Article Title: Nanobody-Based Lateral Flow Assay for Rapid Zika Virus Detection

doi: 10.1021/acssynbio.4c00819

Figure Lengend Snippet: Binding analysis of ZIKV_Nb32 and ZIKV_NbD6 to the Zika NS1 antigen using BLI. Data were fit with a 1:1 binding model. (A) GST-tagged nanobodies immobilized on anti-GST sensors were exposed to a 2-fold dilution series of Zika NS1 starting at 100 nM. (B) NS1 target immobilized on Ni-NTA sensors exposed to the same GST-tagged nanobodies provided in the solution. The 2-fold dilution series started from 100 nM (Nb32) or 12.5 nM (NbD6). (C) Ni-NTA-immobilized NS1 exposed to StrepTag-II versions of both nanobodies using the same dilutions as in (B). The 13 nM trace was excluded as an outlier from the analysis of Nb32 (not shown). Structures of nanobodies are based on AlphaFold3 models. The NS1 tetramer and GST structures were taken from PDB ( 8WBG and 1GNW , respectively).

Article Snippet: The ZIKV SPH2015 NS1 target protein was purchased from Sinobiological (cat: 40544-V07H, Figure S7 ).

Techniques: Binding Assay

Journal: ACS Synthetic Biology

Article Title: Nanobody-Based Lateral Flow Assay for Rapid Zika Virus Detection

doi: 10.1021/acssynbio.4c00819

Figure Lengend Snippet: Characterization of Zika NS1 by mass photometry. (A) The contrast distribution of Zika NS1 single molecule collisions at varying concentrations (25, 50, 100, and 250 nM) indicated the presence of oligomeric species at higher concentrations. (B) Apparent molecular weights (±1 SD) deduced from contrast distribution peaks in A are plotted against NS1 (monomer) concentration ( M r of monomer = 43.5 kDa). The expected M r for different oligomers (monomer M r = 43.5 kDa) are shown as broken lines with shading indicating the average standard deviation (expected accuracy) from contrast distribution peaks in that size range.

Article Snippet: The ZIKV SPH2015 NS1 target protein was purchased from Sinobiological (cat: 40544-V07H, Figure S7 ).

Techniques: Concentration Assay, Standard Deviation

Journal: ACS Synthetic Biology

Article Title: Nanobody-Based Lateral Flow Assay for Rapid Zika Virus Detection

doi: 10.1021/acssynbio.4c00819

Figure Lengend Snippet: Competitive BLI assay for (A) binding of NbD6 alone (replicated from Figure C) and (B) binding of NbD6 after sensor saturation with Nb32. Results were fit to a 1:1 binding model. Zika NS1 was immobilized on Ni-NTA sensors as before. NbD6 was applied in varying concentrations (2-fold serial dilution starting from 100 nM). (C) representative sensor trace (50 nM NbD6) showing all the steps in the competitive assay. Structures of nanobodies are based on AlphaFold3 models. The NS1 tetramer and GST structures were taken from PDB ( 8WBG and 1GNW , respectively).

Article Snippet: The ZIKV SPH2015 NS1 target protein was purchased from Sinobiological (cat: 40544-V07H, Figure S7 ).

Techniques: Binding Assay, Serial Dilution

Journal: ACS Synthetic Biology

Article Title: Nanobody-Based Lateral Flow Assay for Rapid Zika Virus Detection

doi: 10.1021/acssynbio.4c00819

Figure Lengend Snippet: LOD determination of ZIKV LFA in running buffer. Lane1–7: dilution series of ZIKV NS1 protein. Lane 1: 2.5 μg/mL, lane 2: 250 ng/mL, lane 3: 25 ng/mL, lane 4: 2.5 ng/mL, lane 5: 0.25 ng/mL. (A) Capture Nb: Nb32-Strep, detection Nb: NbD6-Strep. NC membrane blocked with 0.6% BSA. (B) Capture Nb: GST-Nb32, detection Nb: NbD6-Strep. NC membrane blocked with 0.6% BSA.

Article Snippet: The ZIKV SPH2015 NS1 target protein was purchased from Sinobiological (cat: 40544-V07H, Figure S7 ).

Techniques: Membrane

Journal: ACS Synthetic Biology

Article Title: Nanobody-Based Lateral Flow Assay for Rapid Zika Virus Detection

doi: 10.1021/acssynbio.4c00819

Figure Lengend Snippet: Validation of ZIKV LFA using Strep-tagged nanobodies in human serum (A) and bovine urine (B). Capture Nb: Nb32-Strep, detection Nb: NbD6-Strep. NC membrane blocked with 0.6% BSA. Lane1–8: dilution series of ZIKV NS1 protein. Lane 1: 2.5 μg/mL, lane 2: 0.5 μg/mL, lane 3: 0.1 μg/mL, lane 4: 20 ng/mL, lane 5: 4 ng/mL, lane 6: 2 ng/mL, lane 7: 1 ng/mL, lane 8: 0.5 ng/mL.

Article Snippet: The ZIKV SPH2015 NS1 target protein was purchased from Sinobiological (cat: 40544-V07H, Figure S7 ).

Techniques: Membrane

Journal: ACS Synthetic Biology

Article Title: Nanobody-Based Lateral Flow Assay for Rapid Zika Virus Detection

doi: 10.1021/acssynbio.4c00819

Figure Lengend Snippet: Dengue NS1 test in our LFA using Strep-tagged nanobodies. Capture Nb: Nb32-Strep, detection Nb: NbD6-Strep. NC membrane blocked with 0.6% BSA. Sample 1: 8.5 μg/mL Dengue serotype 1 NS1 protein, sample 2: 26.7 μg/mL Dengue serotype 2 NS1 protein, sample 3: 15.6 μg/mL Dengue serotype 3 NS1 protein, sample 4: 11.0 μg/mL Dengue serotype 4 NS1 protein.

Article Snippet: The ZIKV SPH2015 NS1 target protein was purchased from Sinobiological (cat: 40544-V07H, Figure S7 ).

Techniques: Membrane