Direct Plate-Reader Measurement of Nitric Oxide Released from Hypoxic Erythrocytes Flowing through a Microfluidic Device

Abstract

The ability to perform a fluorescence-based quantitative determination of a biologically important analyte directly released from mammalian cells using a standard microtiter plate reader to measure wells integrated into a microfluidic device is reported. Specifically, the amount of nitric oxide (NO) released from flowing erythrocytes (ERYs) exposed to a hypoxic buffer is measured using a fluorescein-based probe. The ERYs are pumped through channels in one layer of the poly(dimethylsiloxane) (PDMS) device; as these cells release NO, it flows through a porous polycarbonate membrane to the probe. The device is then placed into a standard microtiter plate reader for measurement, with the entire calibration and analyte determination occurring simultaneously. Using this method, NO release from hypoxic ERYs was determined to be 6.9 +/- 1.8 microM, a significantly increased value in comparison to that from normoxic ERYs of 0.60 +/- 0.04 microM (p < 0.001, n = 4 rabbits). Furthermore, the reproducibility (reported as a %RSD) of measuring fluorescence standards was 3.5%. Detection limits, dynamic range, and optimal membrane pore diameters are also reported. This device enables the use of a standard high-throughput tool (the plate reader) to measure analytes in a microfluidic device, the ability to improve the quantitative determination of a relatively unstable molecule (NO), and the incorporation of a flow component and blood constituent into a system that can be combined with microtiter plate technology.

Microfluidic device with tunable post arrays and integrated electrodes for studying cellular release

“…Subsequently, 7% RBC samples were prepared and incubated with a oxyrase/buffer solution that has been previously used to make RBC suspensions hypoxic. 27 After the 7% hematocrit RBC sample was incubated with the enzyme solution, they were injected into the cell channel, and their NO release was detected at the glassy carbon-Pt-black-0.05% Nafion electrode. The hypoxic 7% RBCs released 16.5 ± 1.5 µM NO, with the error being expressed as pooled standard deviation (3 donors, each donor run in triplicate).…”

“…This value is almost double previously reported data of hypoxic RBC-NO release using the aforementioned sandwich fluidic device containing a polycarbonate membrane to allow sampling and using fluorescence detection (after derivatization with DAF-FM). 27 The values can be reasoned in that we achieve close to real-time detection, where with fluorescence detection, the incubation period (30 min) and multidimensional chip design may lead to NO degradation or possibly absorption of NO into the PDMS and/or polycarbonate membrane. NO production is inhibited with L-NAME, a competitive inhibitor of nitric oxide synthase (NOS).…”

“…It has been previously shown that this 30 minute incubation period reduces the dissolved oxygen in a buffer to approximately 3% of the saturated concentration. 27 For inhibition studies with L-N G -Nitroarginine methyl ester hydrochloride (L-NAME, Sigma-Aldrich), the RBC samples were incubated in 100 µM L-NAME at room temperature for 1 hour prior to the addition of oxyrase.…”

New Opportunities in Metabolomics and Biochemical Phenotyping for Plant Systems Biology

“…Besides involving complex designs and fabrication techniques prohibiting widespread use due to cost and/or time for production, microfluidic systems may require unfamiliar laboratory habits. Therefore, one logical next step is the integration with the standardized microplate layout, thus taking advantage of the extensive work of the lab automation community (Choi & Cunningham, 2007;Halpin & Spence, 2010). Strikingly, such integration might ultimately result in methodologies enabling high density analyses on very large numbers of samples, thus breaking the relationship depicted in Figure 1.…”

Integrated hybrid polystyrene–polydimethylsiloxane device for monitoring cellular release with microchip electrophoresis and electrochemical detection

“…A recent study demonstrated the effectiveness of integrating flowing cells, a porous polycarbonate membrane, and a plate reader for measurement of nitric oxide released from red blood cells. 4 On-chip red blood cell lysis and electrochemical analysis of intracellular glutathione have also been integrated in a microchip format. 5 The incorporation of a microchip-based separation has also been demonstrated using both off- and on-chip sampling techniques.…”