Cells were resuspended in the DEP buffer at an OD600 of ca. 0.05 and quickly introduced into the 3DiDEP microchannel via the fluidic reservoirs (see section S2 for details about the 3DiDEP microdevice). A “linear sweep” DC voltage difference increasing linearly with time at 1 V/s from 0 to 100 V was applied across the channel via an HVS-448 high-voltage power supply (LabSmith), controlled by a customized LabVIEW program. The SYTO BC fluorescence intensity increased with time as bacterial cells accumulated near the constricted region (movie S1) and was recorded by time-lapse image sequences captured at 1 frame/s using a CoolSNAP HQ2 cooled charge-coupled device (CCD) camera (Photometrics) fitted to an inverted fluorescence microscope (Nikon). The fluorescent intensity data (background subtracted) near the constriction versus time (i.e., the applied voltage) were fitted into a polyline with two segments, whose intersection point was taken as a variable optimized using the least squares method by a customized MATLAB R2015b (MathWorks) code. The applied voltage corresponding to the determined intersection point of the two segments was extracted as the trapping voltage (Fig. 1E) during 3DiDEP.

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