High-Precision Electrophoretic Mobility Measurements of Single Particles and Red Blood Cells Using High-Voltage Microfluidic Transverse AC Electrophoresis (HV-TrACE).

Single-particle characterization is essential for understanding the behavior and interactions of particles, particularly in biological contexts where changes in physical properties can influence their biological functions. However, current characterization methods face challenges in achieving precise measurements, especially with heterogeneous biological particles. Previously, we reported TrACE, in which the electrodes were integrated into the microchannel. While the device demonstrated excellent performance, we sought to eliminate electrode charging to provide a constant electrophoretic velocity and increase the electrophoretic excursions of the particles. In the HV-TrACE design, the electrodes are removed from the microchannel and placed in reservoirs at the ends of side channels, allowing the use of voltages above the water electrolysis threshold. The particle tracking region is centered in the microchannel cross formed by the side channels, where a transverse electric field is applied. The addition of neutral hydrogel plugs suppresses electroosmotic and hydrodynamic flow through the side channels. When the particle oscillations are driven by a square wave in HV-TrACE, the particle trajectories are linear within each half-period, creating a triangular-shaped trajectory. HV-TrACE improves measurement precision by 1) increasing the amplitude of the particle response, and 2) averaging all the data points within the trajectory. The system's performance is demonstrated using charged polystyrene particles and label-free analysis of red blood cells. With improved precision in the electrophoretic mobility measurements and compatibility with physiological buffers, the HV-TrACE system has great potential for advancing single-particle characterization and single-cell phenotyping.