Fig. 4: Massively parallel dynamic force spectroscopy under multiplexed dynamic loading rates.

A Schematic of FLO-Chip outline depicting serially connected microchannels with varying width. B (i) Representative bright-field image of the FLO-Chip microchannels (250 µm, 500 µm, 750 µm, and 1000 µm in width) coated with beads tethered to the coverslip using DIG-AntiDIG binding interaction. The zoomed-in images of coated FLO-Chip depicting selection of beads that are tethered to the surface via a single tether. The difference between the detected bead center position under backward flow (red solid line) and forward flow (cyan solid line) was used to select tethered beads. (ii) The rupture flow rate for each selected bead was determined by monitoring the bead area when the beads ware subjected to linearly increasing flow rate. The flow rate at which each bead was undetected was marked as the corresponding rupture flow rate. (iii) Probability of the selected beads to remain bound under increasing flow rate. Bead rupture was more probable under smaller flow rates in narrower channels. C Calibration of bead drag force (F) with respect to the inlet average velocity (V) in the 1000 μm wide microchannel. N = 30 beads were monitored over three independent experiments. The calibration line (red dash line) indicated a linear relationship between drag force and average perfusion velocity with slope of ~3.0 ± 0.2 pN s mm⁻¹. D Dynamic force spectroscopy of 9nt DNA unzipping with 78% GC content under four different loading rates tested simultaneously on the same chip. E Dynamic force spectroscopy of (i) DIG-AntiDIG dissociation and (ii) 9nt DNA unzipping tested under a wide range of loading rates (10⁻²−10² pN.s⁻¹ and 10⁻²−10¹ pN.s⁻¹, respectively). N = 3 most probable rupture forces (F^*) were estimated based on random selection without replication of the recorded rupture force data under each loading rate. The estimated F^* values are presented as mean values ±SD. Source data are provided as a Source Data file.