cover plate. For the two embossed inlet channels, two connecting
capillaries (360-µm o.d., 50-µm i.d., 280 mm long) were inserted
into each channel until 10 mm of each of the two capillaries was
inside the embossed channels. Another connecting capillary (360-
µm o.d., 50-µm i.d., 180 mm long) was inserted into the embossed
outlet channel until 10 mm of the capillary was inside the
embossed channel. Then, the embossed substrate plate (Figure
3B) and the embossed cover plate (Figure 3C) were aligned and
thermally bonded for 10 min. After this thermal bonding process,
epoxy glue was applied to the edges of the bonded chip around
the connecting capillaries to reinforce the bonding in that area.
P reparation of the Monolithic SP E Column. The monomer
and porogen mixture was similar to those reported by others.27-29
A typical formulation of monomer and porogen mixture was as
follows. Five milligrams of DAP and 3 mg of AMPS were dissolved
in 500 µL of methanol and 250 µL of hexane, to which 150 µL of
BMA and 100 µL of EDMA were added. After being purged with
nitrogen for 5 min, the mixture was aspirated into the chip using
a syringe. A mask containing one layer of black tape and one layer
of aluminum foil was placed over the chip with the 25-mm-long
embossed channel in the right side of the chip uncovered. The
polymerization was initiated by UV irradiation for 20 min under
UV lamp (UVL-28, 365 nm, VWR, West Chester, PA). When the
polymerization process was complete, the remaining monomer
mixture inside the connecting capillaries and nonpolymerized
monomer in the chip were removed with methanol. In Figure 3A
is shown a schematic representation of the completed chip with
the integrated monolithic column.
Figure 3. (A) Schematic diagram of the Zeonor polymeric chip (Z)
containing an incorporated SPE monolithic column. C1, C2, inlet
connecting capillaries; C3, outlet connecting capillary; RC, reaction
channels; M, monolithic SPE column. (B) The substrate plate with
embossed reaction channels (RC). (C) The cover plate embossed
against three fused-silica capillaries (EC1, EC2, EC3) to make channels
for connecting capillaries and monolithic SPE column (see text for
further details).
Imipramine is converted to its N-demethylated metabolite
primarily by CYP2C19,22 which may be inhibited by the action of
tranylcypromine.23,24 Tranylcypromine was therefore used as a
chemical inhibitor to determine its IC50 value for the CYP2C19
imipramine N-demethylation-mediated reaction. The concentra-
tions of tranylcypromine (0, 5, 10, and 15 µM) used for the
calculation of the IC50 were chosen according to those previously
shown for this compound to inhibit CYP2C19-mediated reac-
tions.25,26 Tranylcypromine was dissolved in 50% acetonitrile (final
concentration of organic solvent inside the chip was e0.5% v/ v)
and was premixed off-chip with the substrate. For the determi-
nation of the IC50, the following equation was used to determine
the formation of metabolite: Am/ Am+Ad (where Am and Ad are
the area of the metabolite’s and the area of the parent drug’s SRM
ion current, respectively). Comparison was made to experimental
control incubations (0 µM inhibitor) and the enzyme activity
expressed as the percentage of control remaining. The IC50 value
was determined by graphical linear interpolation.
Integration of a P orous Monolithic SP E Column on the
Chip. Figure 3A shows the layout of the polymer microfluidic chip
containing an incorporated monolithic SPE column. Zeonor
polymer plate was cut into 4 cm by 6 cm pieces. The substrate
plate was embossed against an etched silicon wafer master to
obtain reaction channels (Figure 3B). The cover plate was then
embossed against three conventional fused-silica capillaries (360-
µm o.d., 50-µm i.d.) as shown in Figure 3C. After completing this
embossing process, the three capillaries were peeled off and three
corresponding embossed channels (18 mm long for the two inlet
channels, 35 mm long for the outlet channel) were left in this
Using the chip with the integrated monolithic column, the P450
enzymatic reaction was performed as described above using 10
µM imipramine as the parent drug. A control experiment was also
performed following the same procedure described above but
using inactivated human liver microsomes.
RESULTS AND DISCUSSION
This work focused on demostrating the feasibility of performing
P450 enzymatic reactions using human liver microsomes in a chip-
based format coupled with ESI-MS detection. The chip was
designed to permit the mixing of the reagents, notably the enzyme
and substrate, by a molecular diffusion process in a pressure-
driven laminar flow. Experiments using a fluorescent substrate
were performed to confirm the mixing process under a fluorescent
microscope (data not shown). A pressure-driven flow inside the
chip was selected because the reactions of P450 enzymes take
place in a complex environment (two membrane-bound protein
components, cofactor, MgCl2). Although electrokinetic driven flow
is easy to implement and is used in most of the current on-chip
enzymatic reactions, it is also sensitive to the physicochemical
properties of the fluid being pumped (pH, ionic strength, viscosity)
and to bubbles in the channel, and it is surface sensitive. To ensure
adequate mixing and preclude these problems, a syringe pump
was used to provide pressure-driven flow.
(22) Madsen, H.; Rasmussen, B. B.; Brosen, K. Clin. Pharmacol. Ther. 1 9 9 7 ,
61, 319-324.
(23) Bu, H. Z.; Knuth, K.; Magis, L.; Teitelbaum, P. J. Pharm. Biomed. Anal.
Enzymatic Reaction. In vitro metabolism using the described
system was demonstrated by the transformation of a mixture of
2 0 0 1 , 25, 437-442.
(24) Hartter, S.; Tybring, G.; Friedberg, T.; Weigmann, H.; Hiemke, C. Pharm.
Res. 2 0 0 2 , 19, 1034-1037.
(25) Yu, C.; Shin, Y. G.; Kosmeder, J. W.; Pezzuto, J. M.; van Breemen, R. B.
Rapid Commun. Mass Spectrom. 2 0 0 3 , 17, 307-313.
(26) Dierks, E. A.; Stams, K. S.; Lim, H.; Cornelius, G.; Zhang, H.; Ball, S. E.
Drug Metab. Dispos. 2 0 0 1 , 29, 23-29.
(27) Yu, C.; Davey, M. H.; Svec, F.; Frechet, J. M. J. Anal. Chem. 2 0 0 1 , 73,
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(29) Tan, A.; Benetton, S.; Henion, J., Anal. Chem., submitted.
Analytical Chemistry, Vol. 75, No. 23, December 1, 2003 6433