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

Abstract

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.

Red blood cells in type 1 diabetes and multiple sclerosis and technologies to measure their emerging roles

“…The gaseous NO diffuses into the vascular smooth muscle cell, resulting in cyclic guanosine monophosphate (cGMP) production and downstream vessel dilation [ [80] , [81] , [82] ]. Numerous reports have confirmed the relationship between RBC-derived ATP release and endothelium-derived NO stimulation/vasodilation [ 7 , [86] , [87] , [88] , [89] , [90] ]. From a beneficial standpoint, the NO produced by endothelial cells has vasoprotective and anti-atherosclerotic effects upon release from the cell and is a factor in blood pressure due to its effect on vessel relaxation [ 83 ].…”

“…Plugs of ATP stimulated endothelial release of NO, which was detected on-chip by a Nafion-coated carbon ink electrode. Building upon work that showed the sensitivity of NO detection in PDMS/polystyrene microchip devices could be increased by using array electrodes modified with platinum black [ 207 ], Townsend et al utilized a parallel channel design that cultured endothelial cells in one part of the device, with RBCs being pumped over the endothelial monolayer to truly mimic blood flowing through a vessel [ 89 ]. They were able to show that RBC-derived ATP could be used to stimulate NO release from the endothelial cells and that upregulation of ATP release from RBCs likewise increased endothelial NO production.…”

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“…Alexandra et al used SERS to examine and evaluate the number and distribution of Silver Nanoparticles in living cervical cancer cells, combined with cell viability measurements [3]. The system schematic of this chapter is shown in Fig.…”

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“…18,19 It can be used to detect very small amounts of liquid samples, such as 10 −9 -10 −18 L. 20 Micro-uidics technology is characterized by its small volume, rapid analysis speed, automatic completion of the entire process of sample analysis, and increasing integration scale, allowing for the development of high-cost, compact, and one-time detection instruments. [21][22][23] Currently, many effective detection technologies have been validated in microuidic devices, including electrochemical methods, 24 uorescence detection methods, 25 and RF sensing methods. 26 For example, Xu et al detected tumor-derived exosomes using a magnetic microuidic chip; 27 Peng et al used online uorescence derivatization ow injection in a microuidic device for the detection of Cr(III) and Cr(VI) in water samples aer solid-phase extraction; 28 Liu et al used a microuidic RF biosensor to monitor cell growth.…”