Analytical Chemistry Chemical Cytometry Quantitates Superoxide

simulations, 34,35 which can be helpful in the design of controlling
flow channels that punctually provide H2O2 solution to micropumps
according to a predetermined schedule.
Autonomous Sequential Switching of the Micropumps.
We next studied the function of the micropump. After the lower
compartment of the pump was filled with H2O2 solution, the
diaphragm inflated and the solution in the upper reservoir was
ejected and then mobilized in the extending flow channel.
During this step, the H2O2 solution moved forward in the
controlling flow channel and filled the lower compartment of
the next pump. From that point forward, the same step was
repeated. Needless to say, the concentration of H2O2 injected
into the controlling flow channel affects the flow velocity of
the solution in the upper flow channel. Although the flow
velocity definitely depends on the H2O2 concentration, significant
leakage and destruction of the gel layer was observed over
a certain threshold, which also depended on the size of the
pump. Considering these dynamics, 1.6 or 3.2 M H2O2 was used
in the following experiments, depending on the size of the
pump.
For the handling of many solutions, several micropumps can
be connected to the controlling flow channel and to the upper
flow channels for solutions to be transported. To adjust the timing
and order of the injection of solutions, the distance between pumps
can be adjusted in a network of flow channels. Compartments and
constrictions can also be introduced, as was discussed earlier. In
the device shown in parts A and B of Figure 4, micropumps are
connected with one another by rectangular compartments at their
sides and centers, respectively. In the device shown in Figure
4B, the compartments are filled with solution after the compartment
of a neighboring micropump in the upper stream is filled
with solution. With the small compartments in the crowded layout,
however, the extension of the liquid columns from the upper
reservoirs was not so significant compared with that of the device
shown in Figure 4A. The device shown in Figure 4C has additional
small compartments to delay the movement of the solution. Note
the difference in the length of the liquid columns, which shows
that the actuation of the micropump can be distinctly delayed in
the lower stream of the controlling flow channel, unlike the device
shown in Figure 4A and 4B. This result demonstrates that
additional delaying structures can in fact be used to adjust the
timing to trigger the actuation of the micropumps.
Coordinated Operation of Micropumps for Chemical
Analyses. By properly designing a network of flow channels with
micropumps located in appropriate positions, microfluidic devices
can be constructed for various purposes, including chemical
analyses that require the specific processing of solutions. In the
device shown in Figure 5, there are two micropumps to eject
solutions and two pumps to apply pressure. Here, H 2O2 was also
used as an analyte. A solution containing H2O2 and Amplex
Red (5 mM) and another solution containing 50 U/mL HRP,
both prepared with a 50 mM Tris-HCl buffer solution (pH 7.4),
were used to fill the reservoirs. After another H2O2 solution
was injected into the controlling flow channel, it first filled the
compartments of the micropumps. Following this, the analyte
H2O2 solution in the reservoir was injected into the upper flow
(34) Erickson, D.; Li, D.; Park, C. B. J. Colloid Interface Sci. 2002, 250, 422–
430.
(35) Young, W.-B. Colloids Surf., A 2004, 234, 123–128.
6874 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
Figure 4. Arrays of micropumps with controlling flow channels with
different structures. Rectangular compartments are connected with
the controlling flow channels at the edges (A), at the center (B), and
via smaller delaying compartments (C).
channels for transport after 9 s (Figure 5B, panels 1 and 2).
After a time delay (117 s), the larger pumps that apply pressure
were switched on and the solutions in the upper flow channel
were pinched off from the rest of the solutions, transported to
the center, and merged in the mixing channel (Figure 5B, panels
3 and 4). The average flow velocity in the mixing channel was 94
µm/s. Solutions containing the enzyme and the substrates were
transported and mixed in the flow channel. The enzymatic reaction
by HRP produced highly fluorescent resorufin, which generated
red fluorescence under a fluorescence microscope. Figure 5C
shows the dependence of the fluorescence intensity on the
concentration. The fluorescence intensity was measured 3 min
after mixing. In the graph plotted on the semilog scale, the
dependence of the fluorescence intensity on the concentration
could be clearly observed.