Microfluidic Device Using a Gold Pillar Array and Integrated Electrodes for On‐chip Endothelial Cell Immobilization, Direct RBC Contact, and Amperometric Detection of Nitric Oxide

We describe a microfluidic device that can be used to detect interactions between red blood cells (RBCs) and endothelial cells using a gold pillar array (created by electrodeposition) and an integrated detection electrode. Endothelial cells can release nitric oxide (NO) via stimulation by RBC-derived ATP. These studies incorporate on-chip endothelial cell immobilization, direct RBC contact, and detection of NO in a single microfluidic device. In order to study the RBC-EC interactions, this work used a microfluidic device made of a PDMS chip with two adjacent channels and a polystyrene base with embedded electrodes for creating a membrane (via gold pillars) and detecting NO (at a glassy carbon electrode coated with platinum-black and Nafion). RBCs were pharmacologically treated with treprostinil in the absence and presence of glybenclamide, and ATP release was determined as was the resultant NO release from endothelial cells. Treprostinil treatment of RBCs resulted in ATP release that stimulated endothelial cells to release on average 1.8 +/- 0.2 nM NO per endothelial cell (average +/- SEM, n=8). Pretreatment of RBCs with glybenclamide inhibited treprostinil-induced ATP release and, therefore, less NO was produced by the endothelial cells (0.92 +/- 0.1 nM NO per endothelial cell, n=7). In the future, this device can be used to study interactions between many other cell types (both adherent and non-adherent cell lines) and incorporate other detection schemes.