Red Blood Cell Behavior within the Exclusion Zone

Abstract

Various hydrophilic polymers have been reported to induce the formation of an exclusion zone (EZ) at their surface, which is devoid of particles and may extend to several hundred microns. It has previously been suggested that even cells may be excluded from the vicinity of the gel, thus raising the possibility of developing microscale cell-separation technologies. Here we explored the behavior of red blood cells (RBCs) in the vicinity of Nafion with the aim of devising a cell-separation method and of understanding the microscale mechanisms of EZ formation. We assembled a PDMS-based microfluidic device housing a 1-mm diameter, 50-micron thick cylinder of Nafion. A suspension of RBCs, prepared from anti-coagulated whole human blood by repeated washing/resuspension in PBS, was infused in the device. The position, shape and spectral properties of RBCs were followed with brightfield video microscopy. Contrary to expectations, RBCs were not excluded from Nafion surface. Rather, a three-phase process of aggregation, lysis and discoloration propagated gradually across the stationary RBC suspension from the Nafion surface towards distal regions. During the discoloration phase RBCs turned brown, pointing at the possibility of acid-hematin formation. Microscpectroscopy measurements supported this hypothesis. Thus, the vicinity of fresh Nafion surface is a highly acidic environment. The spatial and temporal propagation of the process suggests that protons diffuse out of the polymer. Soaking Nafion extensively in PBS resulted in the disappearance of the RBC-associated phenomenon, indicating that the thermodynamic driving force of particle exclusion is most likely the presence of a steep proton gradient between Nafion and the surrounding buffer solution. Due to its high acidity, unequilibrated Nafion has limited applications in cell-separation methodologies.