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Enzymatic degradation of p-chlorophenol in a two-phase flow

microchannel system

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Tatsuo Maruyama,a Jun-ichi Uchida,a Tomohiro Ohkawa,b Toru Futami,b Koji

Katayama,b Kei-ichiro Nishizawa,b Ken-ichiro Sotowa,a Fukiko Kubota,a Noriho
Kamiyaa and Masahiro Goto*a
a

Department of Applied Chemistry, Graduate School of Engineering, Kyushu University,

6-10-1 Hakozaki, Fukuoka 812-8581, Japan
b Tokyo Research Laboratories, Tosoh Corporation, 2743-1 Hayakawa, Ayase, Kanagawa
252-1123, Japan
Received 19th August 2003, Accepted 26th September 2003
First published as an Advance Article on the web 14th October 2003

Enzymatic degradation of p-chlorophenol was carried out in a two-phase flow in a microchannel (100 mm width,
25 mm depth) fabricated on a glass plate (70 mm 3 38 mm). This is the first report on the enzymatic reaction in a
two-phase flow on a microfluidic device. The surface of the microchannel was partially modified with
octadecylsilane groups to be hydrophobic, thus allowing clear phase separation at the end-junction of the
microchannel. The enzyme (laccase), which is surface active, was solubilized in a succinic aqueous buffer and the
substrate (p-chlorophenol) was in isooctane. The degradation of p-chlorophenol occurred mainly at the
aqueousorganic interface in the microchannel. We investigated the effects of flow velocity and microchannel
shape on the enzymatic degradation of p-chlorophenol. Assuming that diffusion of the substrate (p-chlorophenol)
is the rate-limiting step in the enzymatic degradation of p-chlorophenol in the microchannel, we proposed a simple
theoretical model for the degradation in the microchannel. The calculated degradation values agreed well with the
experimental data.

Introduction
Miniaturization of chemical reaction devices for the development of effective, space- and energy-saving devices has
attracted much interest. In the last decade, many researchers
have described chemical and biochemical reactions involving
electroosmotic pumping on a microchip. A chromatographic
system involved with a biological reaction on a microchip that
employed electroosmotic pumping was reported by Harrison et
al. (1996),1 and Wang et al. (2001)2 developed an enzyme
immunoassay system on a glass microchip. The electroosmoticdriven flow is, however, applicable only for the conducting
fluid, not for organic solvents. Pressure-driven liquid manipulation has recently come to be employed in microfluidic devices
instead of electroosmotic pumping, because the pressure-driven
systems, usually based on syringe pumping, can control all
kinds of liquids. Tokeshi et al. (2000) succeeded in solvent
extraction of a metal ion on a glass microchip for the first time
using a syringe pump.3 Wenn et al. developed a mesh
microreactor for two-phase (liquidliquid or gasliquid) reactions and discussed the kinetics of the two-phase reactions in the
microreactor.4
Due to the miniaturization, the sample volumes applied in a
microfluidic device are very small compared with those of
conventional bulk reactors, which prevent on-chip detection
using a UV-vis spectrophotometer. Therefore, many research
papers on microfluidic devices have involved laser-induced
fluorescence for monitoring the molecular behavior or chemical
reactions. Fluorescent measurement, however, limits the application of the microfluidic device to fluorescent chemistry.5,6
Collection of reacted medium from a microfluidic device allows
the use of conventional analyses such as spectroscopy, but the
chemical reactions should not progress outside the microfluidic
device during or after sample collection.
Biochemical reactions are expected to be suitable for
microfluidic devices, because the biomolecules such as en308

zymes and antibodies are expensive, and the amounts available

are often limited. Several research groups have studied
enzymatic reactions in microfluidic devices, demonstrating the
advantages of the enzyme reaction in such a device.710 There
is, however, no report on enzymatic reaction in a two-phase
(liquidliquid) flow system in a microfluidic device. Enzymatic
reactions in a heterogeneous two-phase system only proceed at
the liquidliquid interface or in the enzyme solution. If the
organic phase containing substrates can be separated and
collected from the enzyme solution in the microfluidic device,
the conversion of the substrate in the organic phase can be
measured outside the device. This type of a microfluidic device
could be connected with conventional or novel miniaturized
analytical apparatus, providing a powerful tool for analyses in
environmental and biomedical fields.
In the present study, we established the separation and
collection of each phase from a two-phase flow in a microfluidic
device by modifying the partial surface of a microchannel,
which allows out-of-chip analysis. Laccase-catalyzed degradation of p-chlorophenol (Scheme 1) in the two-phase flow in a
microchannel was then investigated, and compared with that of
a batchwise reaction. We proposed a simple theoretical model
for the mass transport between the two phases in the
microchannel and obtained good agreement with the experimental results.

Scheme 1 Dehalogenation of p-chlorophenol catalyzed by laccase

Lab Chip, 2003, 3, 308312

DOI: 10.1039/b309982b
This journal is The Royal Society of Chemistry 2003

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Materials and methods

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Materials
Laccase from Trametes sp. (MW 62 kDa, pI 3.0), which is a
lignin peroxidase, was kindly supplied from Daiwa Kasei, Co.,
Ltd. Trichloro(octadecyl)silane was purchased from Aldrich.
All the other chemicals were purchased from Wako Pure
Chemicals, Osaka.
The microchannel was fabricated by a photolithographic wet
etching method11 on a pyrex glass plate (38 mm 3 70 mm). The
glass plate was initially coated with a 20 nm Cr layer and then
with a 100 nm Au layer. A positive photoresist was spin-coated
on the Au layer. UV light was exposed through a photomask
using a mask aligner to transfer the microchannel pattern onto
the photoresist. The Cr and Au layers were etched with
Ce(NH4)2(NO3)6 and I2/NH4I solutions, respectively. The bare
glass surface was etched with a HF solution. The remaining Cr
and Au layers were thoroughly removed in I2/NH4I and
Ce(NH4)2(NO3)6 solutions. Inlet and outlet holes were mechanically drilled on another pyrex glass plate (cover glass
plate). The microfabricated glass plate was covered by the cover
glass plate and thermally laminated at 650 C. Fig. 1 shows the
layout and dimensions of the microchannel fabricated on a glass
plate. The confluent part of the microchannel was typically 100
mm wide and 25 mm deep. The inlet and outlet channels were 50
mm wide and 25 mm deep.
The surface of the outlet channel (for an organic phase) was
chemically modified with an octadecylsilane group. A toluene
solution containing 10 vol% of trichloro(octadecyl)silane and
pure toluene were fed in parallel from each outlet. The flow
rates of both solutions were 0.5 mL h21 for 2 h. The flow in the
microchannel was laminar, so that the trichloro(octadecyl)silane solution tended to flow on one side of the confluent
microchannel, resulting in the chemical modification of one
side of the confluent microchannel. After the chemical
modification, the microchannels were continuously washed

Fig. 1 Microfluidic device and reaction system used in the present

study.

with pure toluene. Thus one of the two channels between the
end-junction and the outlets was chemically modified to be
hydrophobic.
Laccase-catalyzed degradation of p-chlorophenol in the
microchannel was carried out as follows: an isooctane solution
containing 100 mmol L21 of p-chlorophenol was fed from the
organic-phase inlet and an aqueous solution (0.05 M succinic
buffer, pH 5.0) containing 1.86 g L21 of laccase was fed from
the aqueous-phase inlet using syringes and syringe pumps
(IC3210, KD Scientific, PA). Both phases were collected from
their respective outlets, and the concentration of p-chlorophenol
in each phase was determined by HPLC (LC-10AT, Shimadzu)
equipped with an ODS column (4 3 250 mm, GLScience)
eluted with 80% acetonitrile in water (0.1% phosphoric acid) at
a flow rate of 1.0 mL min21 using a UV detector at 282 nm.
The same reaction was also carried out in a screw-cap bottle
using 1 mL of both the isooctane and the aqueous solutions,
which were the same as the solutions employed in the
microchannel experiment.
The interfacial tension between isooctane and the succinic
buffer solution containing laccase was measured at 25 C using
a drop volume tensiometer (Lauda TVT2S, Germany).
All the experiments were carried out at least in duplicate and
values given are the average of the results.

Results and discussion

Formation of a two-phase flow, and phase separation in
the microchannel
Fig. 2a shows the two-phase (waterisooctane) flow in a 20 cm
long microchannel. The aqueous phase was dyed using orange
G. The stable two-phase flow and the clear liquidliquid
interface obviously formed over the entire microchannel. When
using the microchannel without any surface modification, the
aqueous flow invaded the upper channel for the organic phase at
the end-junction of the microchannel. The naked glass surface
was hydrophilic and likely to be wetted by water, which
explains the invasion of the aqueous phase. On the other hand,

Fig. 2 Photographs of the two-phase flow in a microchannel.

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Fig. 2b shows the two-phase flow formed in the microchannel

modified by trichloro(octadecyl)silane. Neither the organic
flow nor aqueous flow invaded the opposite channel at the endjunction of the microchannel with partial hydrophobic modification and each phase was collected from each channel
without contamination by another phase. Hibara et al. also
reported that the octadecylsilane modification of a microchannel allowed stable phase confluence and separation in a
microchannel.12 These results showed that the surface hydrophobicity of the microchannel plays a critical role in the phase
separation in a microfluidic device.
Laccase-catalyzed degradation of p-chlorophenol in the
microchannel
Using the partially hydrophobic microchannel, the enzymatic
degradation of p-chlorophenol catalyzed by laccase in a twophase flow was carried out with varying flow velocities, from
0.045 to 1.8 ml h21. The substrate, p-chlorophenol, was initially
dissolved in the organic phase. The contact between the organic
and aqueous flows in the microchannel started the diffusion of
p-chlorophenol into the aqueous phase, and the p-chlorophenol
was enzymatically degraded at the waterisooctane interface.
The transfer of p-chlorophenol and the degradation at the
interface occurred only in the confluent part of the microchannel. The conversion of p-chlorophenol is plotted in Fig. 3.
As expected, the degradation of p-chlorophenol decreased with
an increase in the flow velocity. The increase in the flow
velocity reduced the contact time between the aqueous and
organic phases, which affected the degradation of p-chlorophenol.

active and spontaneously adsorb onto the liquidliquid interface. In order to determine the surface property of laccase, we
investigated the interfacial tension between isooctane and a
succinic buffer by using the various laccase concentrations from
0 to 1.86 g L21 (Fig. 4). The interfacial tension gradually
decreased with increasing laccase concentration. This result
suggests that a laccase molecule is surface active and adsorbs
onto the isooctanesuccinic buffer interface. In the present
study, we employed laccase at 1.86 g L21 for the enzymatic
degradation of p-chlorophenol in a microchannel. The measurement of the interfacial tension suggests that laccase molecules
adsorb at the aqueousorganic interface in the microchannel and
are highly concentrated at this interface. Therefore, the laccasecatalyzed reaction was thought to occur mainly at the aqueous
organic interface.
Comparison of the reaction in the microchannel with that
in a batchwise reactor

Enzymatic reactions in a two-phase system often occur at the

aqueousorganic interface, because many enzymes are surface

One of the advantages of a microfluidic device as a reactor is the

large relative interface area. In particular, an interface-based
reaction such as the one in the present study would benefit
greatly from the employment of a microfluidic device. In the
work reported here we investigated the enzymatic degradation
of p-chlorophenol in a screw-cap bottle and compared the
results with those obtained in the microfluidic device. The
reaction efficiency (Jm, defined as the conversion of pchlorophenol divided by the aqueousorganic interface area and
time) based on the organic-aqueous interface area in each
reactor system is summarized in Table 1. We attempted three
reactions with a screw-cap bottle as a reactor. One was a stable
two-phase system without shaking, which provided only 0.3%
of the Jm obtained by the microfluidic device, as there was no
mixing of the reaction medium, and a long diffusion distance for
the substrate (the depth of each phase was 1.2 cm). The second
also consisted of a stable two-phase system but with gentle

Fig. 3 Effect of flow velocity on the conversion of p-chlorophenol.

Fig. 4 Interfacial tension at the isooctanewater interface.

Effect of laccase on interfacial tension between isooctane

and succinic buffer

Table 1 Enzymatic degradation of p-chlorophenol in a batchwise reactor and a microfluidic device

Reaction system

Jm a/mol m22s21

Interface area/m2

Specific
interface are/m21

8.2 3 1026
7.5 3 1027
2.0 3 103
2.7 3 1028
8.1 3 1025
8.1 3 10
1.2 3 1026
8.1 3 1025
8.1 3 10
0.7 3 1028
Not determined
Not determined
a Jm was defined as the conversion of p-chlorophenol divided by the aqueous-organic interface area and time.Specific interface area was defined as interface
are divided by volume.c The flow velocity of the organic phase was 0.5 mL h21. The laccase concentration was 1.86 g L21. d The reaction medium was gently
shaken by a mechanical shaker for 30 min. e The reaction medium composed of the organic and aqueous phases was vigorously shaken by a magnetic stirrer
bar for 30 min. The total interface area between the organic and aqueous phases could not be determined.

Two-phase flow in the microfluidic device (100 3 25 mm) c

Two-phase system in a screw-cap bottle without shaking
Two-phase system in a screw-cap bottle with gentle shaking d
Emulsified medium in a screw-cap bottle with vigorous stirring e

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shaking. The gentle shaking did not cause emulsification and

kept the two phases stable. The gentle shaking induced
recirculation flow in the organic phase, thereby ensuring
sufficient diffusion of p-chlorophenol molecules to the interface, and the Jm achieved was up to 50 times higher than that of
the two-phase system without shaking, but lower than that
achieved by the microfluidic device. The third reaction
consisted of an emulsified system caused by vigorous stirring
with a magnetic stirrer bar. The emulsification provided a large
interface between the isooctane and aqueous solutions. The Jm
obtained in the emulsified reaction medium was, however,
much lower than that of the two-phase system without shaking.
It has been reported that some enzymes are denatured and
inactivated by the shear stress of stirring or by an oilwater
interface.13,14 The emulsified reaction system reported here also
probably induced the denaturation of laccase, which resulted in
the much lower Jm. These results suggest that the microfluidic
device produces a high reaction efficiency in the reaction
involving a liquidliquid interface, especially when using an
interface-sensitive enzyme.

where J(x) is the flux of p-chlorophenol, D is the diffusion

coefficient of p-chlorophenol in isooctane, C is the concentration of p-chlorophenol, and x is the distance from the aqueous
organic interface. The mass balance of p-chlorophenol provides
the following equation:
(2)
where t is the contact time between the aqueous and organic
phases in the microchannel. Using the boundary condition that
C(0,t) is always zero, eqns (1) and (2) produce
(3)
where C0 is the initial p-chlorophenol concentration (100 mM).
Thus the concentration F(t) of p-chlorophenol at contact time t
in the microchannel can be written as
(4)

Theoretical model for the reaction in a microchannel

The Reynolds number of the organic-phase flow at the highest
flow rate (3 mL h21) was lower than 50, meaning that all the
flow tested in the microchannel were laminar. We then
proposed a simple theoretical model for predicting the reaction,
with the following assumptions: the distribution of flow
velocity in a microchannel could be ignored, the p-chlorophenol
diffused at the interface was immediately degraded by laccase
because of the high laccase concentration,15 and the diffusion of
p-chlorophenol was a rate-limiting step in the reaction. A one
dimensional diffusion model (Fig. 5a) was established by using
Ficks law:
(1)

where d is the width of the organic phase (typically 50 mm). The

final conversion R(t) of p-chlorophenol at the end of the
microchannel can be expressed as
(5)
The diffusion coefficient D of p-chlorophenol at 25 C was
calculated to be 1.77 m2 s21 by using the WilkeChang
equation.16 Fig. 5b depicts the conversion of p-chlorophenol
calculated with eqn. (5), and the experimental data as a function
of contact time. This simple theoretical model provides a good
agreement between the calculated values and the experimental
data, which suggests that other chemical phenomena in
microfluidic devices could also be easily predictable.

Effect of the microchannel shape on p-chlorophenol

conversion
The degradation of p-chlorophenol was carried out in microchannels of various dimensions, namely 100 3 25, 70 3 25, 150
3 25, 100 3 10 and 100 3 40 mm. The two-phase flows in
microchannels of dimensions 70 3 25 and 100 3 10 mm were
more unstable than those in the other dimensions of microchannel. This indicates that the glass surface and the aspect ratio
of the microchannel are essential for obtaining stable two-phase
flow. Table 2 summarizes the comparison between the
experimental data and the calculated values. In each case, the
contact time between the organic and aqueous phases was set to
0.15 s. The conversion of p-chlorophenol increased with
increasing relative interface area ([organicaqueous interface

Table 2 Effect of channel shape on the degradation of p-chlorophenol

Fig. 5 Illustration of the microchannel for the diffusion model (a) and
comparison of the calculated p-chlorophenol conversion (solid line) with
the experimental data (filled circles) (b).

Microchannel
dimension
(mm 3 mm)

Relative interface
area a [m21]

100 3 25
70 3 25
150 3 25
100 3 10
100 3 40

1.0 3 104
1.4 3 104
6.7 3 103
1.0 3 104
1.0 3 104

Experimental
value b (%)

Calculated
value (%)

34
36
50
48
25
25
36
36
43
36
a The relative interface area was defined as the aqueousorganic interface
area divided by the microchannel volume. b The contact time between the
organic and aqueous phases was kept constant at 0.15 s.

Lab Chip, 2003, 3, 308312

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area]/[microchannel volume]). The experimental values also

agreed well with the calculated values.

References
1

Published on 14 October 2003. Downloaded by National University of Singapore on 02/11/2016 05:58:24.

Conclusions

In the present study, we carried out an enzymatic reaction in a

two-phase flow microchannel system for the first time. We
found that the surface properties of the microchannel played an
essential role in the behavior of the two-phase flow, and that
control of the surface properties succeeded in the clear
separation of the two phases in the microchannel. Our simple
theoretical model for the reaction in the microfluidic device
provides a good agreement with the experimental data, which
suggests that mass transfer in a microfluidic device could be
easily described by fluid mechanics.

4
5
6
7
8
9
10
11

Acknowledgements

12
13

This research was supported by a Grant-in-Aid for Science

Research (No. 15560656) from the Ministry of Education,
Science Sports & Culture of Japan. We would like to thank
Daiwa Kasei Co., Ltd (Osaka, Japan) for generously providing
laccase.

14
15
16

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D. J. Harrison, K. Fluri, N. Chiem, T. Tang and Z. H. Fan, Sens.

Actuators, B, 1996, 33, 105109.
J. Wang, A. Ibanez, M. P. Chatrathi and A. Escarpa, Anal. Chem.,
2001, 73, 53235327.
M. Tokeshi, T. Minagawa and T. Kitamori, Anal. Chem., 2000, 72,
17111714.
D. A. Wenn, J. E. A. Shaw and B. Mackenzie, Lab Chip, 2003, 3,
180186.
G. J. M. Bruin, Electrophoresis, 2000, 21, 39313951.
E. Verpoorte, Electrophoresis, 2002, 23, 677712.
Y. Tanaka, M. N. Slyadnev, A. Hibara, M. Tokeshi and T. Kitamori,
J. Chromatogr., A, 2000, 894, 4551.
E. LHostis, P. E. Michel, G. C. Fiaccabrino, D. J. Strike, N. F. de
Rooij and M. Koudelka-Hep, Sens. Actuators, B, 2000, 64,
156162.
E. Eteshola and D. Leckband, Sens. Actuators, B, 2001, 72,
129133.
M. Miyazaki, H. Nakamura and H. Maeda, Chem. Lett., 2001,
442443.
A. Hibara, M. Tokeshi, K. Uchiyama, H. Hisamoto and T. Kitamori,
Anal. Sci., 2001, 17, 8994.
A. Hibara, M. Nonaka, H. Hisamoto, K. Uchiyama, Y. Kikutani, M.
Tokeshi and T. Kitamori, Anal. Chem., 2002, 74, 17241728.
J. M. Cassells and P. J. Halling, Enzyme Microb. Technol., 1990, 12,
755759.
A. S. Ghatorae, M. J. Guerra, G. Bell and P. J. Halling, Biotechnol.
Bioeng., 1994, 44, 13551361.
J. Michizoe, K. Shinohara, T. Maruyama, K. Kusakabe, H. Maeda
and M. Goto, Kagaku Kogaku Ronbunshu (in Japanese), 2003, 29,
8286.
C. R. Wilke and P. Chang, AIChE J., 1955, 1, 264.