A 3D-printed, multi-modal microfluidic device for measuring nitric oxide and ATP release from flowing red blood cells.

In this paper, a 3D-printed multi-modal device was designed and fabricated to simultaneously detect nitric oxide (NO) and adenosine triphosphate (ATP) in red blood cell suspensions prepared from whole blood. Once a sample was injected into the device, NO was first detected (via amperometry) using a three-electrode, dual-opposed, electrode configuration with a platinum-black/Nafion coated gold working electrode. After in-line amperometric detection of NO, ATP was detected via a chemiluminescence reaction, with a luciferin/luciferase solution continuously pumped into an integrated mixing T and the resulting light being measured with a PMT underneath the channel. The device was optimized for mixing/reaction conditions, limits of detection (40 nM for NO and 30 nM for ATP), and sensitivity. This device was used to determine the basal (normoxic) levels of NO and ATP in red blood cells, as well as an increase in concentration of both analytes under hypoxic conditions. Finally, the effect of storing red blood cells in a commonly used storage solution was also investigated by monitoring the production of NO and ATP over a three-week storage time.

3D-Printed Microfluidic Device with In-line Amperometric Detection that Also Enables Multi-Modal Detection.

Microfluidic amperometric detectors often include a reservoir to house auxiliary and reference electrodes, making subsequent detection downstream challenging. Here, we present an in-line microfluidic device with amperometric detection that incorporates a three-electrode set-up, made possible by threading electrodes into a 3D-printed flow cell. The electrodes consist of a commercially available threaded reference electrode and electrodes fabricated in commercially available fittings. This approach centers the working electrode in the fluidic channel enabling the use of a pillar working electrode that is shown to increase sensitivity, as compared to a traditional thin-layer electrode. In addition, the working and auxiliary electrodes can be directly opposed, with this configuration leading to a more uniform potential being applied to the working electrode as well as fewer issues with any iR drop. To demonstrate the ability to incorporate a separate mode of detection downstream from the electrochemical flow cell, the device is modified to include a mixing T for introduction of reagents for chemiluminescent detection of ATP (via the luciferin-luciferase reaction), leading to a single 3D-printed device that can be used to detect norepinephrine and ATP, nearly simultaneously, by amperometry and chemiluminescence, respectively. This approach opens numerous possibilities, where microfluidics with in-line amperometry can be used in continuous circulation studies or in conjunction with other downstream detection events to study complex systems.

An Interfacial Sodium Ion is an Essential Structural Feature of Fluc Family Fluoride Channels.

Fluc family fluoride channels are assembled as primitive antiparallel homodimers. Crystallographic studies revealed a cation bound at the center of the protein, where it is coordinated at the dimer interface by main chain carbonyl oxygen atoms from the midmembrane breaks in two corresponding transmembrane helices. Here, we show that this cation is a stably bound sodium ion, and although it is not a transported substrate, its presence is required for the channel to adopt an open, fluoride-conducting conformation. The interfacial site is selective for sodium over other cations, except for Li+, which competes with Na+ for binding, but does not support channel activity. The strictly structural role fulfilled by this sodium provides new context to understand the structures, mechanisms, and evolutionary origins of widespread Na+-coupled transporters.

PolyJet 3D-Printed Enclosed Microfluidic Channels without Photocurable Supports.

Microfluidic devices have historically been prepared using fabrication techniques that often include photolithography and/or etching. Recently, additive manufacturing technologies, commonly known as 3D-printing, have emerged as fabrication tools for microfluidic devices. Unfortunately, PolyJet 3D-printing, which utilizes a photocurable resin that can be accurately printed, requires the use of support material for any designed void space internal to the model. Removing the support material from the printed channels is difficult in small channels with single dimensions of less than ∼200 µm and nearly impossible to remove from designs that contain turns or serpentines. Here, we describe techniques for printing channels ranging in cross sections from 0.6 cm × 1.5 cm to 125 µm × 54 µm utilizing commercially available PolyJet printers that require minimal to no postprocessing to form sealed channels. Specifically, printer software manipulation allows printing of one model with an open channel or void that is sealed with either a viscous liquid or a polycarbonate membrane (no commercially available support material). The printer stage is then adjusted and a second model is printed directly on top of the first model with the selected support system. Both the liquid-fill and the membrane method have enough structural integrity to support the printing resin while it is being cured. Importantly, such complex channel geometries as serpentine and Y-mixers can be designed, printed, and in use in under 2 h. We demonstrate device utility by measuring ATP release from flowing red blood cells using a luciferin/luciferase chemiluminescent assay that involves on-chip mixing and optical detection.

Insert-based microfluidics for 3D cell culture with analysis.

We present an insert-based approach to fabricate scalable and multiplexable microfluidic devices for 3D cell culture and integration with downstream detection modules. Laser-cut inserts with a layer of electrospun fibers are used as a scaffold for 3D cell culture, with the inserts being easily assembled in a 3D-printed fluidic device for flow-based studies. With this approach, the number and types of cells (on the inserts) in one fluidic device can be customized. Moreover, after an investigation (i.e., stimulation) under flowing conditions, the cell-laden inserts can be removed easily for subsequent studies including imaging and cell lysis. In this paper, we first discuss the fabrication of the device and characterization of the fibrous inserts. Two device designs containing two (channel width = 260 µm) and four (channel width = 180 µm) inserts, respectively, were used for different experiments in this study. Cell adhesion on the inserts with flowing media through the device was tested by culturing endothelial cells. Macrophages were cultured and stimulated under different conditions, the results of which indicate that the fibrous scaffolds under flow conditions result in dramatic effects on the amount and kinetics of TNF-α production (after LPS stimulation). Finally, we show that the cell module can be integrated with a downstream absorbance detection scheme. Overall, this technology represents a new and versatile way to culture cells in a more in vivo fashion for in vitro studies with online detection modules. Graphical abstract This paper describes an insert-based microfluidic device for 3D cell culture that can be easily scaled, multiplexed, and integrated with downstream analytical modules.