Journal: bioRxiv

Article Title: DNA Hanger: Surface Free Single-Molecule Blotting Platform

doi: 10.1101/2024.10.23.619861

Figure Lengend Snippet: (a) Schematic of the biotinylated DNA hanger. Biotins were generated by photo-coupling reagent (Photobiotin), which introduces biotin-group onto the bases on nucleic acids upon 405 nm irradiation. A 3’-digoxigenin oligonucleotide was ligated to 12 nt cohesive ends of the linearized λ-phage DNA. (b) Due to the 3D-etched quartz surface, the excitation laser forms a thin light sheet near the barrier where the biotinylated DNA is tethered via a streptavidin and anti-digoxigenin antibody interaction. Target molecules (5’-biotin-ssDNA-Cy5) are immobilized on the DNA Hanger through the interaction between streptavidin and biotin. (c) Schematic of DNA Hanger (upper left) and representative images with Cy5-streptavidin (upper right). Schematic of DNA Hanger imaging with unlabeled streptavidin and Cy5-labeled DNA (bottom left), along with representative images of the DNA Hanger displaying various concentrations of Cy5 labeled DNA (bottom right).

Article Snippet: To achieve the attachment, a solution of the modified λ -phage DNA molecules (20 pM, diluted in 1x TE buffer, pH 7.4) was injected into the flow channel at a flow rate of 0.07 ml/min with a syringe pump (Harvard Apparatus).

Techniques: Generated, Irradiation, Imaging, Labeling

Journal: bioRxiv

Article Title: DNA Hanger: Surface Free Single-Molecule Blotting Platform

doi: 10.1101/2024.10.23.619861

Figure Lengend Snippet: (a) Schematic of TNF-α and its antibodies (cAb or dAb) attached to the DNA Hanger. The cAb, conjugated with AF647 and biotin, is immobilized on λ-phage DNA through the biotin-streptavidin interaction, and captures TNF-α, which is detected by a fluorescently labeled dAb. (b) The nonspecific binding of dAb or TNF-α to the DNA Hanger or cAb. Each diamond dot indicates the average of the number of dAb per DNA in a field of view. A total 10,784 DNA molecules (left), 9,954 (middle), and 6,894 (right) across 30 fields of view were analyzed, respectively. P- values were calculated using one-way ANOVA test ( ****P < 0.00005). “ns” indicates no statistically significant difference ( P ≥ 0.05). (c) Representative fluorescent images of cAb exhibiting DNA molecules and dAb binding to TNF-α across different concentrations, ranging from 0 pM to 100 pM. (d) The number of dAb per specific length (10 μm) of DNA was calculated at various concentrations. A total of 3,121 DNA molecules in 18 fields of view (0 pM), 3,465 in 17 fields of view (3 pM), 2,173 in 16 fields of view (10 pM), and 1,699 in 18 fields of view (30 pM) were analyzed, respectively. All data were collected from at least three independent experiments.

Article Snippet: To achieve the attachment, a solution of the modified λ -phage DNA molecules (20 pM, diluted in 1x TE buffer, pH 7.4) was injected into the flow channel at a flow rate of 0.07 ml/min with a syringe pump (Harvard Apparatus).

Techniques: Labeling, Binding Assay