The surface charge of red blood cells (RBCs), commonly expressed as zeta potential, is a key biophysical parameter influencing RBC aggregation, deformability, and microcirculatory behavior. Despite its physiological and clinical relevance, routine assessment of RBC zeta potential is limited by methodological complexity and poor reproducibility of conventional microelectrophoretic techniques. In this study, we present an automated method for determining the zeta potential of individual RBCs based on computer-assisted tracking of cell motion during microelectrophoresis. Video recordings of RBC movement in an electric field were analyzed using automated spot detection and trajectory reconstruction, allowing electrophoretic mobility and zeta potential to be calculated at the single-cell level. Application of the method to RBCs from healthy donors revealed reproducible zeta potential distributions, while additionally revealing distinct subpopulations of cells with markedly reduced surface charge. The effect of electric field direction on zeta potential was systematically evaluated, demonstrating the contribution of diffuse layer relaxation effects and supporting the use of bidirectional measurements to improve accuracy. Furthermore, incubation of RBCs with gramicidin S, trypsin, and neuraminidase induced pronounced and statistically significant reductions in zeta potential, confirming the sensitivity of the method to alterations in membrane composition and sialic acid content. Overall, the proposed automated microelectrophoretic approach enables objective, high-resolution analysis of RBC electrokinetic properties and overcomes key limitations of manual techniques. This method holds promise for studying red blood cell aging, membrane damage, and drug–cell interactions, and may contribute to the development of novel biophysical biomarkers relevant to hematological and systemic disorders.
Borikov et al. (Sun,) studied this question.