Poster: A 3D-Printed Fluidic Device for Concurrent Detection of Secreted Signaling Molecules from Hypoxic Red Blood Cells

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Poster presentation at SLAS2014.

Jayda Erkal, B.A. in Chemistry
Research Assistant, Michigan State University


In response to stimuli such as hypoxia (low oxygen tension), red blood cells (RBCs) release various metabolites including adenosine triphosphate (ATP) and nitric oxide (NO), which have been implicated in mechanisms of vasodilation. A rugged, reusable platform that allows for control and detection of oxygen levels in a flowing stream of RBCs as well as concurrent measurement of these signaling molecules is vital to studies probing RBC response to hypoxia. Many studies to elucidate signaling molecule release from RBCs are conducted on microfluidic-based platforms, which are commonly fabricated from polydimethyl siloxane or other soft polymers. The reproducibility between such devices as well as physical limitations of device construction due to lack of material rigidity limits the device designs that can be fabricated, and ultimately, the innovative potential of and control over an elegant experimental design. In this regard, 3D printing allows more control over the geometric design of devices, even considering the yet unexplored minimum printable feature dimensions. We present a 3D-printed device, based on the commercial 96-well plate, which is compatible with a commercial plate reader and contains 12 - 0.5 mm x 0.5 mm channels to enable sample flow. Transwell polyester membrane inserts with 0.4 μm pores were inserted into pre-engineered wells, which allows for the membrane to be in contact with the channel and samples during flow. More importantly, the device allows one to distinguish molecules that are RBC-derived from molecules released by other cell types such as endothelial cells, which is necessary when conducting mechanistic studies of vasodilation. The well inserts were loaded with buffer for ATP measurements or 20 μM of the extracellular NO probe DAF-FM (ex: 488 nm, em: 520 nm) for NO measurements. Standards, consisting of NO and ATP in buffer, or samples, consisting of 7% RBCs, were pumped with a syringe pump through microbore capillary tubing at 6 μL/min and through the device for 20 minutes. ATP and NO released from flowing RBCs diffuse across the polyester membrane and into the detection well. To measure the quantity of ATP released from the RBCs, an aliquot of luciferin-luciferase was added to the sample and chemiluminescence was measured with a commercial plate reader. To detect the quantity of NO released from RBCs, DAF-FM fluorescence was read using the commercial plate reader directly from each well insert. Up to 12 samples or standards can be flowed at one time, rendering this device one with high throughout potential. The integration of the 3D printed multichannel device with oxygen and NO sensing electrodes will also be presented.