Simple and Convenient Three-Electrode Layout for Real-Time Electrorotation Measurement Upon Chemical Stimulation

We developed a novel electrorotation (ROT) device featuring a microwell array with three electrodes. This device allows to monitor the increase in membrane capacitance of cells subjected to chemical stimulation. The microwell array is integrated into the bottom of a fluidic channel and holds rotating cells during stimulation with a solution containing a chemical agent. Positive dielectrophoresis (p-DEP) effectively traps cells in microwells, whereas negative DEP (n-DEP) facilitates the rapid formation of single-cell presence. Alternating current (AC) voltages with a 120° phase shift applied across the three electrodes enable vertical and simultaneous rotation of cells. We observed a peak in rotation rate as a function of applied frequency, with the frequency spectrum shifting to lower frequencies as membrane capacitance increased. A positive correlation was identified between rotation rate and membrane capacitance, so monitoring in the low-frequency range is advantageous. Although n-DEP at lower frequencies risks removing cells from microwells, the continuous monitoring of the ROT rate during chemical stimulation was achieved by regulating the height of the ROT center of cells. We demonstrated the monitoring of membrane capacitance increase induced by Ca²⁺ influx from ionomycin. This simple configuration facilitates statistical analysis of ROT rates without fluorescent labeling, making it suitable for label-free assessments of white blood cells' responses to stimuli.