Extended Data Fig. 4: Determination of DNA probe density on Au nanoparticles surface.
From: A reversible shearing DNA probe for visualizing mechanically strong receptors in living cells
To calibrate the surface density of the DNA tension sensor, we used the method described by Wang and Ha et al.23 to plot the relationship between the fluorescence grayscale units of the sample (mean intensity) and the DNA probe density. First, we incubated aqueous solutions containing 25 nM, 12.5 nM, and 6.25 nM Cy3B-unfolded DNA probe (a scrambled DNA hairpin probe annealing with a complementary strand, the structure was shown in a) on the AuNPs-modified coverslips for 30 minutes, then washed off the unbinding probes and measured the average fluorescence intensity of glass as a function of the incubation concertation of the Cy3B-unfolded DNA probe (b). There is a linear relationship between the mean fluorescence intensity of the surface and the DNA incubation concentration within this concentration range (c). By extrapolation, incubation of 0.05 nM Cy3B-unfolded DNA probe on the surface should correspond to mean fluorescence intensity of 14.4. we also found a molecular density of 0.149 molecules/μm2 on the surface (0.05 nM) using a single-molecule TIRF image (b). Taken together, 96.6 units in mean fluorescence intensity on the Cy3B-unfolded DNA probe surface is equivalent to 1 molecule/ μm2. Given that the average QE of the Cy3B-folded DNA probe is 98.3% (Fig. 1 in main text), the 1.6 units mean fluorescence intensity on the RSDTP-Cy3B coated surface should be equivalent to 1 molecule/ μm2. We could also plot a calibration curve between intensity and molecular density for RSDTP (Cy3B) coated surface, as shown in d. This calibration curve allows us to determine RSDTP (Cy3B) surface density by measuring the average intensity of a coverslip. For example, the 45-pN RSDTP coated surface’s molecular density was estimated to be 671 molecules/μm2 (e).
