Direct Plate-Reader Measurement of Nitric Oxide
Released from Hypoxic Erythrocytes Flowing
through a Microfluidic Device
Stephen T. Halpin and Dana M. Spence*
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824
The ability to perform a fluorescence-based quantitative
determination of a biologically important analyte directly
released from mammalian cells using a standard micro-
titer 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 fluo-
rescence 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 quantita-
tive 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 systems for biological analyses, especially cellular
analyses, is a research area that has greatly expanded.1-7
Microfluidic-based systems offer many desirable features for
cellular analyses including fast analysis times (on the order of
seconds if direct detection is used), the possibility for mass
production, small channel volumes (on the order of nanoliters),
and the ability to inject and manipulate sample volumes as small
as picoliters.
Traditionally, these devices are fabricated in glass8 or other
materials such as plastics, low temperature ceramics, and poly-
(dimethylsiloxane) (PDMS).9 PDMS has gained widespread use
in cellular analyses due to the ease of fabrication and the fact that
PDMS is gas permeable and can be reversibly sealed to a variety
of substrates.10 Micron-sized channels are made by soft lithog-
raphy techniques, where a “master” of a positive relief structure
is first fabricated on a silicon wafer and then a mixture of PDMS
prepolymer/curing agent is poured against the master. Important
to this work, our group11,12 and others13-15 have shown that
PDMS can be used to create 3-dimensional fluidic devices. Initial
works showed that PDMS-based devices can be used to pattern
both proteins and cells, and because PDMS is permeable to gases,
the cells behave normally.16-18 It has been shown that using
PDMS devices to culture cells, allows not only patterning of cells
but also delivery of agents to the adhered cells and monitoring of
events that occur over select cells or a portion of a cell.18 More
recent work has been aimed at operations such as cell culture,2,19
cell sorting and handling,20 and single cell analysis.7,21-23
* To whom correspondence should be addressed. E-mail: dspence@
chemistry.msu.edu.
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Anal. Chem. 2010, 82, 7492–7497
10.1021/ac101130s  2010 American Chemical Society7492 Analytical Chemistry, Vol. 82, No. 17, September 1, 2010
Published on Web 08/03/2010

Despite these successes, use of microfluidic devices for
biological analyses has not become mainstream and there are
several possible reasons for this. First, many of the reported chip
systems involve complicated fabrication, thereby prohibiting
widespread use due to cost and/or time for production. Another
problem in the field of microfluidic-based cell analyses is the
demonstration of cell culture alone and a lack of integrating
analysis steps to measure intra- or extra-cellular molecules or
pathways once cells are ready for analysis. Finally, most biomedi-
cal researchers are familiar with the use of microtiter plates, well
readers, fluid handlers, etc. that are based on 96-well plate
technology. Often, it is difficult to couple microchip systems to
such technology in high use by the biomedical fields.
Continuous demands for high throughput analyses, largely
spurred by drug discovery, have created an industry centered on
the 96-well plate and equipment for handling these plates in an
automated manner. In order to more accurately test drug
candidates, an in vitro system which more closely represents in
vivo conditions (e.g., incorporation of blood flow, interactions of
multiple cell types, real-time measurement of the analytes of
interest) should be more useful in preventing false conclusions
in the drug development process. There has been other progress
toward automating analyses by combining high throughput
technologies with microfluidic devices. For example, much work
has been done in the area of purifying and sequencing DNA, such
as radial systems developed for separation and sequencing that
can perform 96 analyses at once,24 and others that can provide
sample-in, answer-out capabilities for DNA analysis.25 Advances
have been made in utilizing existing high throughput technologies
implementing solid phase purification of DNA from blood using
polycarbonate devices.26-28 Other attempts at observing enzyme
kinetics utilizing absorbance measurements29 and surface plasmon
resonance-based detection have been successfully performed.30
Recent attempts by our lab31 to mimic certain characteristics
of blood vessels while performing analysis in a high throughput
manner have shown that multiple analyte detection in the presence
of erythrocytes (ERYs) is possible utilizing a fluorescence mac-
rostereomicroscope with a CCD camera as a detector; however,
these studies were limited in throughput by the imaging area of
the microscope. Furthermore, the wells of the device were set
up in a format that did not replicate those of the 96-well plate.
Here, we present results from studies designed to develop a
microfluidic device that utilizes a microplate reader as a detector
to analyze the nitric oxide release (NO) from hypoxic ERYs. In
the device described here, the NO that is released from the ERYs
is able to immediately (limited by diffusion rate of the gaseous
NO to the probe above the porous membrane) react with a
fluorescent probe, thus resulting in a quantitative determination
of NO that is closer to the time point at which it was actually
released from the ERY.
EXPERIMENTAL SECTION
Preparation of ERYs. ERYs used in this study were obtained
from animals following protocols approved by the Animal Inves-
tigation Committee at Michigan State University. Male New
Zealand white rabbits (2.0-2.5 kg) were anesthetized using
ketamine (8 mL/kg, im) and xylazine (1 mg/kg, im) followed by
pentobarbital sodium (15 mg/kg, iv). Rabbits were ventilated with
room air at a rate of 20 breaths/min using a tidal volume of 20
mL/kg by placing a cannula in the trachea. A catheter was then
placed into the carotid artery for administration of heparin (500
units, iv) prior to exsanguination through the same catheter.
Approximately 80 mL of whole blood is collected from each animal.
Whole blood was then centrifuged three times at 500g at 25 °C
for 10 min. After each centrifugation, the plasma and buffy coat
were collected for other experimentation before the remaining
solution was resuspended and washed twice in a physiological
salt solution (PSS) (containing in mM, 4.7 KCl, 2.0 CaCl2, 140.5
NaCl, 12 MgSO4, 21.0 tris(hydroxymethyl)aminomethane, and
5.6 glucose with 5% bovine serum albumin at a final pH of 7.4).
All samples were prepared and analyzed within 8 h of harvest-
ing from the animal.
Fabrication of PDMS Arrays. In this device, PDMS slabs
were fabricated using the established techniques of soft lithog-
raphy.32,33 Briefly, masters were fabricated by spin coating piranha
cleaned silicon wafers with SU-8 50 photoresist (Microchem
Corporation, Newton MA) at 500 rpm for 15 s and then 1000 rpm
for 30 s, producing features that were 100 µm tall. Caution:
Piranha solutions are extremely corrosive. The coated wafer is then
exposed, through a transparency mask containing 200 µm chan-
nels, to ultraviolet light to induce cross-linking of the photoresist,
producing the master after development. The completed device,
as described pictorially in Figure 1, consisted of two slabs of
PDMS, with a 0.2 µm pore diameter polycarbonate membrane
(Steriltech Inc., Kent, WA) sealed between the layers. While other
groups34,35 have used PDMS membranes in NO sensing, here,
we employ polycarbonate membranes due to success involving
cell culture on polycarbonate, and the potential future utility to
detect multiple analytes12 that may not be able to permeate
silicone-based membranes. To facilitate the thermocuring of the
completed device, a 20:1 ratio of bulk polymer to curing agent of
Sylgard 184 (Ellsworth Adhesives, Germantown, WI) was used
on surfaces to be sealed to the membranes or PDMS. A 5:1
mixture of bulk polymer to curing agent was then used to coat
this surface, adding rigidity to the device. Inlets to the 200 µm
wide by 100 µm tall channels were punched using 20 gauge tubing,
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7493Analytical Chemistry, Vol. 82, No. 17, September 1, 2010