Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 3. Electrochemical detection experimental setup.
ments, the microdialysis probe outlet will be connected to the
glucose sample inlet of the device using a simple, low-deadvolume
tubing connection.) For the comparison of system
response to premixed glucose-GOx solutions and direct reaction
of glucose and GOx in the channel, premixed solution was
prepared using a 1:3 volume ratio of 5.6 mM glucose and 150
U/mL GOx in a vial. After adding the two solutions, the vial
was gently shaken 10 times and introduced in the reactor
channel through both inlets approximately 5 min later after
shaking. A two-position actuator switching valve (Valco Instrument
Co., Inc.) was adapted to alternatively introduce buffer
solution and glucose sample solution to measure background
and sensing signals, respectively. Once the flow rate had been
set each time, the valve was switched from buffer solution to
sample solution. The same solution was used for measurements
at each flow rate, and current measurements were made
continuously as flow rate was varied. All experiments were
carried out at room temperature, and data were saved directly
on a computer. A schematic diagram of the electrochemical
detection experimental setup is shown in Figure 3.
3. RESULTS AND DISCUSSION
3.1. Characterization of the Micromixers. A fabricated
micromixer was characterized by capturing images at different
distances from the Y-junction using a fluorescence microscope
and determining the variation in fluorescence intensity across the
width of the channel. The standard deviation (SD) was calculated
using the following eq 3: 30
SD ) � 1
N
N ∑ i)1
(x i - x¯) 2
where xi is the gray-scale intensity value of pixel i, and x¯ is the
mean intensity value of pixels across the entire channel.
Quantitative values of the SD of mixing efficiency varied
between 0.5 for completely unmixed solutions (variation of
intensity from 0 for KI solution to 100% fluorescence for
fluorescein) and 0 for completely mixed solutions (intensity
equal across the channel).
Figure 4 shows the SD of intensity versus distance as a function
of flow rate, comparing channels with slanted grooves and no
(30) Xia, H. M.; Wan, S. Y. M.; Shu, C.; Chew, Y. T. Lab Chip 2005, 5, 748–
755.
6760 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
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Figure 4. Comparison of microfluidic mixers having no grooves and
slanted grooves as a function of distance from the Y-junction and at
different flow rates (one device, n ) 3). The flow rate in each case is
the total flow rate in the mixing channel. The flow rates of fluorescein
and KI at the inlets are each half of the total flow rate.
grooves. Flow rates that fall in the range required to achieve high
glucose recoveries in microdialysis sampling of subcutaneous
tissue were considered. The results showed that grooved channels
have a higher mixing efficiency compared to channels without
grooves. In fact, mixing in channels with slanted grooves is almost
complete at a distance of 1 cm along the channel at total flow
rates ranging from 0.4 to 2 µL/min (Reynolds number ) 0.08 at
1 µL/min). This result confirms that grooves formed in the
channel ceiling at an oblique angle with respect to flow direction
enhance the mixing rate of two adjacent solution streams.
Differences in measured standard deviations of fluorescence
intensity at shorter distances along the channel are most likely
due to the reproducibility of the experiment itself. The depth and
width of the grooves were designed based on Stroock’s geometric
parameters (relative groove height to channel height, R < 0.3;
channel height, h E width of the channel, w) and have values of
6-8 and 50 µm, respectively, as shown in Figure 2 (c). 27 These
initial results of mixing characterization were used as important
information for further mixing and reaction experiments.
3.2. Cyclic Voltammograms of Three Different Types of
Microfluidic Channel. Cyclic voltammograms were carried out
to check the electrochemical properties of the microfluidic chip.
A ferri/ferrocyanide couple provides an ideal electrochemical
model for a preliminary chip test with integrated electrodes. The
redox couple ferri/ferrocyanide reaction is as follows (eq 4):
3- - 4-
Fe(CN) 6 + e a Fe(CN)6 E° ) 0.356 V (4)
First, the redox curve was examined with a stationary flow when
1mM(K3Fe(CN)6) was introduced in the channel at a scan
range of -0.3 to 0.7 V. The oxidation and reduction peaks were
observed at 0.21 V (35 nA current) and 0.10 V (56 nA current)
respectively, at a scan rate of 100 mV/s (data not shown). For
cyclic voltammetry under continuous flow conditions, 1 mM
Fe(CN)6 3- /1 mM Fe(CN)6 4- was prepared in 20 mM phosphatebuffered
solution (pH 7.2) containing 0.1 M potassium nitrate
as a supporting electrolyte. Experiments were performed at a