Analytical Chemistry Chemical Cytometry Quantitates Superoxide

down until the water evaporated. The amount of the powder
that remained was between 0.75 and 1.35 mg, depending on
the size of the micropump. To prevent leakage of the powder
and more effectively produce oxygen bubbles and exert
pressure upon the diaphragm, the compartment beneath the
diaphragm was stuffed with a plug of a PVA-SbQ gel, leaving
a space below it that could be filled with the H2O2 solution
introduced from the controlling flow channel. In forming the
plug, that space was filled with a precursor solution of PVA-
SbQ, which was then cured under a UV light. Air vents were
formed at appropriate locations to release pressure and facilitate
the transport and filling of the H2O2 solution. A circular
reservoir (diameter 1.0 mm) for a solution to be transported
was also formed with PDMS on the diaphragm layer and was
connected to a flow channel. The flow channels extending from
the reservoirs of several micropumps formed an appropriate
network that reflected their different purposes. An inlet was
formed on the reservoir to be filled with a transported solution.
Micropumps that were used only to apply pressure were
formed in a similar manner. In this case, an inlet for the
transported solution was not formed. For all devices, the
heights of the controlling flow channels and of the flow
channels in which solutions would be transported were 75 and
150 µm, respectively. The width of the flow channel for
transportation was 500 µm. After the reservoirs in the upper
PDMS substrate were filled with the necessary solutions, the
entire structure was inserted between two poly(methyl methacrylate)
(PMMA) plates and then fixed in place with bolts
and nuts.
Procedure and Principle of Operation. A change in the
volume in the upper solution reservoir is caused by the deformation
of the diaphragm that follows the production of oxygen
bubbles produced by the catalytic decomposition of H2O2. First,
aH2O2 solution is transported in the controlling flow channel
by capillary action and is injected into the lower compartment
of the micropump (Figure 1B, top). When the solution reaches
the MnO2 powder below the diaphragm, oxygen bubbles are
produced by the catalytic decomposition of H2O2:
MnO2 2H2O298 2H 2 O + O 2
The diaphragm then inflates and exerts pressure upon the solution
in the reservoir. As a result, the solution is pushed forward in the
flow channel and is transported to the lower stream (Figure 1B,
bottom). The structure can also be modified so that the reservoir
is filled with only air, instead of a solution to be transported. In
this case, only a change in pressure is generated to move a liquid
column that may be present in the lower stream of the extending
flow channel. Several micropumps can be connected to the
controlling flow channel and the flow channel for the transport of
necessary solutions. Each micropump can be switched on individually
and sequentially according to a predetermined schedule
that is programmed in the shape of the network of controlling
flow channels.
Construction and Operation of Analysis Systems. To
demonstrate the sequential manipulation of solutions for chemical
6872 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
analyses, two devices were fabricated. In one of them (Figure 5),
a solution containing 50 U/mL HRP and another solution containing
H 2O2 as an analyte and Amplex Red (5 mM) were filled in
the reservoir of two micropumps. The solutions were prepared
with a 50 mM Tris-HCl buffer solution (pH 7.4). After the
solutions were ejected from the pumps and merged at the
T-junction according to the programmed pumping, their
intensity of fluorescence was measured using a fluorescence
microscope (VB-G25, Keyence, Tokyo, Japan) equipped with
a CCD detection system (Keyence VB-7000/7010). In a more
complicated device (Figure 6), enzymes (GOD and LOD) were
immobilized in two of three injection ports. HRP was immobilized
in a reaction chamber formed in the lower stream. In the
immobilization of the enzymes, an enzyme solution, a 0.1 wt %
BSA solution, and a 0.1 wt % GA solution were mixed in a 1:1:1
ratio. The mixed solution was then dropped into the corresponding
reservoirs or the reaction chamber (5 µL for the GOD and LOD
solutions and 1 µL for the HRP solution), and a cross-linking
reaction was allowed to proceed. Following this, the enzymeimmobilized
layers were immersed in a 0.1 M glycine solution
for 60 min. The activity of the immobilized enzymes was 1.3 U
for GOD, 0.17 U for LOD, and 3.3 × 10 -2 U for HRP. After the
injection ports were filled with solutions containing either
glucose or lactate or both of them, along with the immobilized
enzymes, H2O2 was produced by the enzymatic reactions. The
solutions were then transported to the reaction chamber. The
enzymatic reactions of HRP were accompanied by the generation
of fluorescence, whose intensity was measured. Values for
time, flow velocity, and fluorescence intensity were obtained
in five measurements, whose averages are used in the
following discussion.
RESULTS AND DISCUSSION
Movement of a Solution in the Controlling Flow Channel.
The controlling flow channel consisted of three walls of PDMS
and a bottom of glass. Although PDMS is hydrophobic (contact
angle 110°), the hydrophilic glass bottom alone (contact angle
15°) was able to generate a sufficient driving force to produce
capillary action. The velocity of the column of H2O2 changed
depending on device parameters such as the width, height, and
wettability of the flow channel. The velocity of a liquid column,
x˙, in a straight flow channel is expressed as follows: 30-33
x˙ ) γLV 8ηx( hw
h + w) 2
[
2 cos θPDMS w
+ cos θPDMS + cos θglass h ]
Here, γLV is the interfacial tension between the solution and
the air, η is the viscosity of the solution, x is the distance from
the inlet of the capillary to the meniscus of the moving liquid
column, h and w are the height and width of the flow channel,
and θPDMS and θglass are the contact angles on PDMS and glass,
respectively.
As could be anticipated from the equation, the movement of
the column slowed as it moved forward in the fabricated flow
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