DNA-Origami Immobilization and Labeling

Flow chambers were assembled by sandwiching double-sided tape in between PEG-passivated glass slide and cover slip with a volume of around 10 µL. Flow chambers contained 1 mm holes at each end for buffer exchange. A tubing with syringe was inserted at one end and sealed with 5-min epoxy. A 20–200 µL pipette tip was inserted at the other end for introducing buffers. First, a 0.1 mg/mL solution of neutravidin (Invitrogen Inc.) in 3% BSA in PBS was introduced into the biotin-PEG/PEG passivated flow cell and incubated for 5 min. Excess neutravidin was washed off with PBS. 250 pM DNA-origami structures were added in 100 µl Buffer B + (5 mM Tris-HCl, 10 mM MgCl₂, 1 mM EDTA and 0.05% (vol/vol) Tween 20 at pH 8.0) per channel, incubated for 15 min and washed with 1 mL buffer C. Gold nanoparticles (Cytodiagnostics, cat: G-90-100) diluted 1:10 in buffer C were introduced and incubated for 5 min before washing with buffer C. The mIgG2a antibody, conjugated with the S1-handle with P3 docking site (S1-P3, Table S2) was incubated at a concentration of 60 nM in 0.5× buffer C + 1.5% BSA in PBS for 15 min. Excess mIgG2a-DNA conjugate was washed off with 2 × 200 µl 0.5x buffer C. For the negative control, instead of the mIgG2a, 60 nM of the S1-P3 handle alone was incubated for 15 min and the following steps remained the same. After washing off the primary probe, the κLC-Nb-7xR3 was added in 0.5x buffer C + 1.5% BSA and incubated for >45 min at RT. Excess κLC-Nb was removed by washing with 2 × 200 µl 0.5× buffer C, 10 min before imaging.