Direct plate-reader measurement of nitric oxide released from hypoxic erythrocytes flowing through a microfluidic device.
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.
Halpin
Stephen T
ST
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Spence
Dana M
DM
D000818:Animals; D015687:Cell Hypoxia; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D004912:Erythrocytes / Q000378:metabolism*; D008297:Male; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D009569:Nitric Oxide / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D004129:Dimethylpolysiloxanes; D009569:Nitric Oxide; C013830:baysilon