Mechanical and Electrical Properties of Red Blood Cells Under Oxidative Stress Using Optical Tweezers

Red blood cells (RBCs) rely on their mechanical and electrostatic properties to maintain microvascular circulation and oxygen delivery. Oxidative stress, a hallmark of many diseases, disrupts these properties by damaging membrane lipids and cytoskeletal proteins. In this study, RBCs from healthy donors were incubated with three oxidative agents, i.e., phenylhydrazine (PHZ), tert-butyl hydroperoxide (t-BHP), and diamide, to investigate how oxidative stress affects the combination of single-cell mechanical, morphological, biochemical, and electrical properties under different oxidizing conditions. All oxidants significantly increased intracellular reactive oxygen species (iROS), with PHZ producing the strongest effect. Morphological analysis revealed acanthocyte and echinocyte formation and a reduced cell diameter. Zeta potential (ζ) remained unchanged across treatments when measured in phosphate-buffered saline (PBS) solution, suggesting a preserved surface charge under these conditions. Mechanical properties were assessed by optical tweezers at the single-cell level to estimate overall elasticity and bending modulus. Untreated and t-BHP-treated RBCs showed comparable elasticity and bending modulus. Diamide increased overall elasticity and slightly elevated bending modulus, while PHZ severely impaired elongation capability, inducing an irregular trapping behavior and prohibiting the calculation of the bending modulus. These findings demonstrate that oxidative stress exerts agent-specific effects on RBC biomechanics, with t-BHP inducing mild alterations, diamide increasing elasticity, and PHZ causing profound damage. To our knowledge, this is the first study to directly correlate oxidative damage, mechanical stiffness, and electric properties across single-cell and population scales, providing a comprehensive evaluation of RBC responses to oxidative injury.