Electrokinetic Analysis of Oxidative Stress in Human Red Blood Cells

Introduction::
Oxidative stress (OS), characterized by an imbalance between the number of reactive oxygen species (ROS) and the cell’s natural antioxidant defense system, inhibits normal cellular function. In red blood cells (RBCs), OS is important as it irreversibly damages the cells, impairs oxygen delivery, and induces RBC aging and hemolysis. OS is found to be a dominant factor in many RBC-related diseases (e.g., sickle cell disease, anemias) and cardiovascular disease. Considering this, a critical need exists for novel, low-cost diagnostic assays for diagnosis and treatment of RBC-related diseases.




Dielectrophoresis (DEP) is an electrokinetic technique which relies on the electrophysiological properties of cells and the suspending media as well as the geometry of the applied electric field to induce movement of these cells. Zeta potential (ζ-potential), is an electrochemical property of cells in suspension typically quantified by an electrophoretic light scattering technique, quantifying macromolecules moving under the influence of an applied electric field. This is used to quantify the electrostatic charge present on the cell surface. There is evidence that ζ-potential and DEP properties, previously considered as independent parameters, may be linked in an interconnected, mechanistic “cell electrome”.




The present work demonstrates DEP and ζ-potential effects on RBCs as a result of oxidative stress. These two measures can help explain how RBCs may modulate their ionic currents during cellular processes. This supports the hypothesis that the RBC electrome, in lieu of a cell nucleus, acts as a controlling mechanism, modulating surface potential through alterations in membrane potential.




Materials and Methods::
RBCs were isolated from human whole blood procured via venous puncture. RBCs were exposed to the OS agent hydrogen peroxide (H₂O₂) at 0-10mM for 2 hours. DEP analysis was conducted on a 3DEP system, providing three DEP cell parameters: membrane conductance (G_EFF), membrane capacitance (C_EFF), and cytoplasm conductivity (σ_CYT). DEP experiments were run for 30 seconds, repeated at least 3 times (n=3) for each experimental group. ζ-potential was measured using Zetasizer Lab (Malvern Analytical) at a maximum of 30 runs per replicate, repeated three times (n=3) for each experimental group. Cells were stained with the Reactive Oxygen Species (ROS) indicator, carboxy-H2DCFDA and analyzed with flow cytometry and under a fluorescent microscope in the FITC spectrum (488nm – 525nm) to monitor ROS for each experimental group RBC morphology was monitored using light microscopy at x20 and x40 magnification. RBCs were also monitored for hemolysis using absorption of 200 µL supernatant samples in a plate reader.




Results, Conclusions, and Discussions::
G_EFF and σ_CYT demonstrated an inverse linear relationship (R2=0.93) with significant differences in G_EFF across all treatments when compared to the 0 mM control and after 1mM dosage for σ_CYT. ζ-potential also decreases magnitude (becomes less negative) significantly after 1mM dosage.




The linear proportionality between G_EFF and σ_CYT is consistent with findings from Hughes et al 2021. Similarly, the observed increase in G_EFF and decrease in ζ-potential is also consistent with previous work showing an overall decrease in DEP force and ζ-potential as a result of OS.Interestingly, the increase in G_EFF at lower dosages compared to σ_CYT and ζ-potential, suggests that a “lag” exists between the membrane becoming less resistive (increased GEFF) and then the decrease in σCYT and ζ-potential. .




The results shown in Figure 1 confirm our hypothesis that ζ-potential and DEP properties are linked in an interconnected, mechanistic “cell electrome”. This also supports our theory that membrane potential is modulating ζ-potential. Further, while the primary known mechanism of action driving the decrease in ζ-potential during RBC OS, is the result of the removal of sialic acid groups by lipid peroxidation, the observed inverse relationship between G_EFF and ζ-potential further suggests that ζ-potential is also modulated by ion flux out of the cell resulting in a change in RBC transmembrane potential. Considering this, future work will seek to determine a direct relationship between transmembrane potential and ζ-potential.






Acknowledgements (Optional): : Funding for this work was supported by Dr. Henslee’s NSF CAREER Award (#2145313) and startup funds from Dr. Henslee in the Wake Forest Department of Engineering. This work was also supported by WFU Engineering Undergraduate research students, Timothy Swimmer, Jessi Dickerson, and Sydney Joseph.


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