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

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  Publication Details (including relevant citation   information):

  Anal. Chem., 2010, 82 (17), pp 7492–7497
  DOI: 10.1021/ac101130s
  Publication Date (Web): August 3, 2010


  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 μM, a significantly increased   value in comparison to that from normoxic ERYs of 0.60 ± 0.04 μM   (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.

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