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. 2022 Aug 25;14(33):3171-3179.
doi: 10.1039/d2ay00931e.

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

Affiliations

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

Elizabeth A Hayter et al. Anal Methods. .

Abstract

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.

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Figures

Figure 1.
Figure 1.
A) CAD rendering of the 3D-printed multi-modal device used in these studies. The device contains threaded ports for fluidic fittings and electrodes. B) The assembled device with threaded working and auxiliary electrodes, a commercially available threaded reference electrode, and threaded fluidic connections. The ATP mixing channel is aligned directly on top of a PMT window for chemiluminescence detection.
Figure 2.
Figure 2.
Characterization of amperometric detection portion of the device for NO detection. A) Micrograph of 100 μm gold working electrode in the printed channel and the same electrode modified with Pt-black and Nafion for selective NO detection. B) Bar graph demonstrating the exclusion of nitrite compared to equimolar injections of NO (95 μM) using the Pt-black/Nafion modified electrode. C) Amperometric trace for NO detection of a 7% RBC sample (triplicate injections, +0.85 V vs. Ag/AgCl). Inset shows trace for injection of 7% RBC sample and an overlay of a 7% RBC sample spiked with 9.5 μM NO.
Figure 3.
Figure 3.
Characterization of mixing in ATP/chemiluminescence portion of the device. A) CAD rendering of the multi-modal, double T device that illustrates the imaging points before and across the mixing channel. B) Graph showing the fluorescence intensity (line scan) across the mixing channel as a function of the distance downstream from the double T intersection. C) Micrographs of fluorescein and RBCs at discrete imaging points before and across the mixing channel (scale bar = 100 μm).
Figure 4.
Figure 4.
Characterization of ATP detection. A) Effect of mixing device (single T vs. double T) on the calibration sensitivity, with a significant increase seen for the double T mixing device. B) Chemiluminescence trace for ATP detection of a 7% RBC sample (triplicate injections). Inset shows trace for injection of 7% RBC sample and an overlay of a 7% RBC sample spiked with 2.5 μM ATP.
Figure 5.
Figure 5.
Use of multi-modal device to quantitate NO and ATP release from healthy RBCs. A) Bar graph showing average NO concentrations from normoxic and hypoxic RBCs (7% solution, n=11 different donations, error = SEM). B) Bar graph showing average ATP concentrations from normoxic and hypoxic RBCs (7% solution, n=10 different donations, error = SEM).
Figure 6.
Figure 6.
Use of multi-modal device to investigate the effect of an approved storage solution on NO and ATP release from a single RBC sample under normoxic and hypoxic conditions over the course of 3 weeks. A) Bar graph showing average NO concentrations from normoxic and hypoxic RBCs (7% solution) as a function of the storage day (stored RBCs were exposed to hypoxic conditions on the day of analysis). B) Bar graph showing average ATP concentrations from normoxic and hypoxic RBCs (7% solution) as a function of the storage day (stored RBCs were exposed to hypoxic conditions on the day of analysis).

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