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