7
MS or matrix-assisted laser desorption ionization (MALDI) MS/
structed by fixing a polyvinylidene fluoride membrane with the
absorbed trypsin inside the capillary fitting for protein digestion.
8
17
MS represents the high-throughput approach for inhibitor screen-
ing of a mixture of compounds using immobilized enzyme
stationary phases. It not only gives the binding constant between
a target enzyme and tested compounds but also provides the
Sakai-Kato et al. fabricated a trypsin-encapsulated enzyme reactor
in the head of the capillary via sol-gel chemistry.18 Yang and co-
workers prepared an encapsulated trypsin enzyme reactor with
titania- and alumina-based sol-gel chemistry on the channel of
microfluidics.19 They also reported a method for preparing a
trypsin reactor on poly(dimethylsiloxane)-based microfluidics
7
inhibition kinetic parameters. Brennan and co-workers prepared
enzyme-doped monolithic capillary-enzyme-reactor columns by
sol-gel chemistry and directly coupled to tandem MS for
9
20
screening of enzyme inhibitors. Surface plasmon resonance (SPR)
using an ionic binding manner. Peterson et al. developed
combined with MS has been developed by Borch and Roepstorff
methods for immobilizing trypsin on the porous polymer mono-
liths. Amankwa and Kuhr directly immobilized the trypsin onto
1
0
21
for enzyme inhibitor screening. In this approach, the enzyme
was immobilized on the sensor chip, and the interactions between
the enzyme and the compounds in the mixture was detected by
SPR. Concurrently, the activity of the immobilized enzyme was
monitored by the coupled MS. Finally, the inhibitor was recovered
from the immobilized enzyme and characterized by MS. In
addition, Leary and co-workers developed a method using MS
combined with immobilized enzyme for high-throughput screening
the inner surface of the capillary via biotin-avidin-biotin technol-
ogy.22 Brennan and co-workers developed sol-gel-derived im-
23
mobilized enzyme microarrays for nanovolumn inhibition assays.
Mao et al. immobilized the streptavidin-conjugated enzyme to the
surface of the microchannels or capillary tubes coated with
24
biotinylated phospholipid bilayers. Zhan et al. reported a method
for fabricating poly(ethylene glycol) hydrogen micropatches
1
1
25
of inhibitors from complex mixtures. Palm and Novotny evalu-
ated PNGase F activity by using an enzyme reactor coupled with
containing enzymes within a channel.
In the present work, we describe a new strategy for the
preparation of the on-column immobilized enzyme microreactor
for rapid screening of enzyme inhibitors via CE. The preparation
of the enzyme microreactor is based on the ionic binding
immobilization approach, which can be easily and automatically
accomplished with the instrument. To demonstrate the feasibility
of our strategy, angiotensin-converting enzyme (ACE) was em-
ployed as an experimental model. The primary function of ACE
is to regulate arterial pressure by converting angiotensin I to
vasoconstrictor angiotensin II. Therefore, ACE is a major target
for developing drugs used to treat hypertension and heart failure,
improve survival following a heart attack, and slow the progression
of kidney disease in people with diabetes.26 The performance and
inhibition kinetics of the immobilized ACE were studied in our
experiments, and the repeatability of the preparation of the enzyme
microreactor was also evaluated. Finally, a small library of
compounds containing 3 commercial ACE inhibitors and 31
natural extracts were tested.
1
2
off-line MALDI-MS. On-line coupling of LC to a continuous-flow
enzyme reactor assayed by MS for screening of enzyme inhibitors
in complex samples has been reported by de Boer et al.13
Capillary electrophoresis (CE) is not only a separation tool with
high separation performance but also a versatile platform for
enzyme study and drug discovery. Among the various CE
techniques, electrophoretically mediated microanalysis (EMMA)
technique is a useful miniature tool for the study of enzymes and
1
4,15
for inhibitor screening.
dramatically improved by using multiplex capillary electrophoresis
The screening throughput can be
16
with UV absorption detection. Alternatively, immobilized enzyme
microreactors fabricated on capillaries and microfluidic chips
represent another promising miniature approach for enzyme
study, peptide mapping in proteomics, and diagnostics. Thus far,
several methods have been reported for the preparation of on-
column enzyme microreactors for capillary electrophoresis (CE),
capillary liquid chromatography, and microfluidic chips. Lee and
co-workers developed a micro trypsin reactor, which was con-
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