Enzymatic processing in microfluidic reactors 405

Biotechnology and Genetic Engineering Reviews - Vol. 25, 405-428 (2008)

Enzymatic Processing in Microfluidic

Reactors

MASAYA MIYAZAKI*1,2, TAKESHI HONDA1#, HIROSHI YAMAGUCHI1,

MARIA PORTIA P. BRIONES1, AND HIDEAKI MAEDA*1,2,3

1
Nanotechnology Research Institute, National Institute of Advanced Industrial
Science and Technology (AIST), Tosu, Saga 841-0052, Japan; 2Department of
Molecular and Material Science, Interdisciplinary Graduate School of Engineering
Sciences, Kyushu University, Kasuga, Fukuoka 816-8580, Japan; 3CREST, Japan
Science and Technology Agency, Kawaguchi, Saitama 332-0012 , Japan

Abstract

Microreaction technology is an interdisciplinary area of science and engineering. It

has attracted the attention of researchers from different fields in the past few years
and consequently, several microreactors have been developed. Enzymes are organic
catalysts used for the production useful substances in an environmentally friendly
way, and have high potential for analytical applications. However, relatively few
enzymatic processes have been commercialized because of problems in the stability of
enzyme molecule, and the cost and efficiency of the reactions. Thus, there have been
demands for innovation in process engineering particularly for enzymatic reactions,
and microreaction devices can serve as efficient tools for the development of enzyme
processes. In this review, we summarize the recent advances of microchannel reaction
technologies and focus our discussion on enzyme microreactors. We discuss the
manufacturing process of microreaction devices and the advantages of microreactors
compared with the conventional reactors. Fundamental techniques for enzyme
microreactors and important applications of this multidisciplinary technology in
chemical processing are also included in our topics.

*To whom correspondence may be addressed (m.miyazaki@aist.go.jp or maeda-h@aist.go.jp)

#Present address: Department of Pharmacology, Yamaguchi University Graduate School of Medicine,
Yamaguchi 755-8505, Japan

Abbreviations: NTA, N,N-bis(carboxymethyl)glycine, which binds Nickel (II) ion, and usually applied
for identification/purification of histidine tag of the engineered enzyme molecule; LIGA, Lithography,
Electroplating, and Molding; PEEK, poly(ether ether ketone).
406 M. Miyazaki et al.

Introduction

Microchannel reaction systems, which can be prepared by microfabrication techniques

or by assembly and modification of microcapillaries, utilize reaction apparatus of small
dimensions (Ehrfeld et al., 2000; Ziaie et al., 2004; Szekeley and Guttman 2005).
These systems take advantage of microfluidics or nanofluidics that enables use of
micro or nanolitre volumes of reactant solutions, and offer high efficiency performance
and repeatability. Therefore, microchannel reaction systems are expected to be the
new and promising technology in the fields of Chemistry, Chemical Engineering and
Biotechnology (Hessel et al., 2004; Watts and Haswell 2005; Chovan and Guttman
2002; Anderson and van den Berg 2003; Cullen et al., 2004; Wang and Holladay
2005; Miyazaki and Maeda 2006). They offer several advantages for performing
chemical reactions over traditional technologies. The key advantages of microsystems
include rapid heat exchange and rapid mass transfer that cannot be achieved by a
conventional batch system. Unlike macro-scale solution, streams of solutions in a
microfluidic system mainly form a laminar flow which allows strict control of reaction
conditions and time. In addition, microchannel reaction systems provide large surface
and interface areas, which are advantageous for many chemical processes such as
extractions and catalytic reactions. Several chemical reaction devices have been
reported to demonstrate their potential applications (Hessel et al., 2004; Watts and
Haswell 2005; Chovan and Guttman 2002; Anderson and van den Berg 2003; Cullen
et al., 2004; Wang and Holladay 2005; Miyazaki and Maeda 2006). Moreover, many
potential applications for miniaturized synthetic reactors require only a small volume
of catalyst.
Enzymatic conversion has recently come to the forefront because of its
environmentally friendly nature. Several enzyme processes have been developed;
however, improvement of the entire process is still required to obtain the benefit
that can be derived from its use and to gain reevaluation as a common or standard
technology (Shoemaker et al., 2003; Garcia-Junceda et al., 2004). Reaction
engineering might provide solutions to develop enzyme reaction processes at the
commercial level (Schmid et al., 2001), and microreaction engineering is one
candidate for such technology. Therefore, several techniques have been developed,
either by solution phase or by immobilizing enzymes, to realize enzyme microreaction
processes (Miyazaki and Maeda 2006; Urban et al., 2006). In this review, we
summarize the recent advances of microchannel reaction technologies especially
on enzyme microreactors. We discuss the manufacturing process of microreaction
devices and the advantages of microreactors compared with conventional reaction
devices. Fundamental techniques underpinning enzyme microreactors and important
applications of this multidisciplinary technology are also presented.

Fundamentals of microreactors

For newcomers of the microfluidic reaction techniques, we would like to start our
review with a brief introduction. Microfluidic reaction occurs in a small space within
the reaction apparatus. Continuous-flow system is mainly employed; in most cases
mechanical pumping, commonly by syringe pumping, or electroosmotic flow, which
is the motion of ions in a solvent environment through very narrow channels, where an
 Enzymatic processing in microfluidic reactors 407

applied potential across the channels cause the ion migration, are used as the driving
force of the reaction system. The microreaction devices that have been developed
so far, can be classified into two types, chip-type microreactors and microcapillary
devices. Chip-type microreactors offer several advantages including easy control
of microfluidics, and integration of many processes into one reaction device. Chip-
type microreactors have been mainly used for the development of bioanalytical
devices. Manufacturing process of such devices are adaptations mainly from the
microelectronics industry. Dry-or wet-etching processes have been used for creating
channels on a silicone or glass plates. Polymer-based materials can be used for
preparation of enzyme microreactors because most of enzyme reactions have been
performed in aqueous solution, especially for bioanalytical use. Polydimethylsiloxane
(PDMS), polymethylmarthacrylate (PMMA), polycarbonate, and Teflon have been
used for preparation of microreaction devices. These plates could be processed by
photolithography, soft lithography, injection molding, embossing, and micromachining
with laser or microdrilling. The LIGA (Lithographie Garbanoforming Abforming; see
Abbreviations) process which consists of a combination of lithography, electrochemical
technology and molding, can also be used for the production of microreactors.
In a microreactor, stable formation of laminar streams of different solutions is
sometimes required although some cases require better mixing by disrupting laminar
streams. Methods for stabilizing multiple laminar flows and micromixing have been
developed. Tokeshi et al developed guide structures at the bottom of microchannel
(Tokeshi et al., 2002). The structures were prepared by wet etching of a glass plate.
A laminar stream of organic solvent and water in this microchannel was stabilized by
these guide structures. Partial surface modification techniques of microchannels has
also been developed. This technique takes advantage of the different surface properties.
Organic solvent prefers hydrophobic regions, whereas aqueous solution goes with
hydrophilic regions. Modification of glass by octadesylsilane was used to stabilize the
flow of organic solvent and aqueous solutions (Hibara et al., 2002). In another study,
UV-sensitive self-assembled monolayer with fluorous chain was used for preparing
partially-modified microchannel surface (Zhao et al., 2002). Our laboratory developed
another method to stabilize solutions (Yamaguchi et al., 2005). Microchannel was
fabricated on both bottom and top plates. Microchannel of one of the two plates was
coated with gold, then treated with alkanethiol to produce a hydrophobic surface. The
resulting microreactor, which forms an upside-down laminar stream, was not only
stabilized by interaction with a surface, but also supported by gravity. Overall, such
partial modification methods are useful for stabilizing laminar streams under pressure
below the critical value. Indeed, the microfluidic phenomenon of laminar flow is one
important aspect in the development of chip-type microreactors.
Micromixers which enhance mixing of two or more different solutions in microspace
have also been constructed. Rapid mixing in microfluidics is difficult to achieve
because under laminar flow mixing of fluids is principally limited to diffusion through
the interface. Several micromixers have been developed by adding devices or materials
in the microchannel, such as electrokinetical mixing (Erickson and Li 2002) and
microbeads (Seong and Crooks 2002). Various types of micromixers which only
require structured microchannels have also been developed. These include a chaotic
mixer with oriented ridges at the bottom of microchannel (Strook et al., 2002), repeated
dividing and merging of fluid (Kim et al., 2002), zig-zag microchannel (Mengeaud et
408 M. Miyazaki et al.

al., 2002), and simple 32 layers in which two solutions are divided into 16 streams and
converge to form 32 layers (Yamaguchi 2003). However, the design and fabrication
of highly efficient micromixers for effective functioning of microfluidic devices are
still desired. The other type of microreaction device consists of a microcapillary. This
is the simplest method which does not require any control of microfluidics, rather,
it uses a microchannel as the reaction space. The major advantage of this type of
microreactor is in scaling up processes which can be achieved by bundling together
microcapillaries. Gas or liquid chromatography parts are mainly used to prepare
this type of microreactor. Capillary type microreactors are mainly used to develop
manufacturing processes, especially catalytic reactions, to take advantage of the large
surface area.

Fundamental techniques for enzyme microreactor in solution-phase

CONTINUOUS-FLOW SOLUTION-PHASE REACTION

Simple micro-enzyme reactions have been performed by solution-phase methods.

Continuous-flow microreaction has been performed on a chip-type microreactor
fabricated from PMMA plate (Miyazaki et al., 2001; Kanno et al., 2002). The reaction
was performed by simple loading of substrate and enzyme solutions into separate
inlets using syringe pumps. Such reactions rely mainly on rapid mass transfer of the
microreaction system. Trypsin-catalyzed hydrolysis of benzoyl-arginine-p-nitroanilide
(Miyazaki et al., 2001) and glycosidase-catalyzed hydrolysis reactions (Kanno et al.,
2002) were performed using this type of microreactor. In all cases, reaction yields
were improved 3 to 5 fold in a microchannel system. These results demonstrate the
possibility of continuous-flow microreaction systems as a tool for further development
of microreaction processes.
Enzyme reaction was also performed in a micromixer format. Glass-bead packed
microreaction systems are mainly employed as micromixing devices (Seong et al.,
2002, Sotowa et al., 2005). In both cases, efficient reaction has been performed.

STOPPED-FLOW REACTION

Microreactors have been examined not only in the continuous-flow mode but also in the
stopped-flow mode of operation. Kitamori and coworkers have described stopped-flow
microreactor devices using glass microchips with Y-shaped channels. The stopped-flow
procedure involves mobilization of reagents through such a device for a designated
period of time using an applied chemical and/or pumped field. The flow is subsequently
paused by the removal of the applied field before re-application of the field. Results
from experiments utilizing the stopped-flow mode have reported an acceleration of
peroxidase-catalyzed reactions (Tanaka et al., 2001)). Such observed increases in the
reaction rates using the stopped-flow technique has been attributed to an effective
increase in residence time within the device corresponding to the different kinetics
associated with these reactions. Stopped-flow microreaction systems with IR heating
has also been developed (Tanaka et al., 2000). The system enables non-contact partial
heating of the reaction solution. Peroxidase-catalyzed reactions have been performed
in a cooled chip equipped with an IR diode laser. The rate of the enzyme reaction
 Enzymatic processing in microfluidic reactors 409

which was initially inhibited due to cooling of the chip to lower the temperature was
increased by non-contact heating by utilizing through photothermal effect produced by
a diode laser. Their findings suggest the possibility of controlling nanoscale reactions
and the precise synthesis of substances by photothermal stimulation.

OTHER SOLUTION-PHASE TECHNIQUES

A multiplex enzyme assay with several simultaneous enzymatic reactions has been
performed in an electrophoretic microreaction device (Xue et al., 2001). The resolving
power of electrophoresis enables several enzyme assays to be analyzed at high speed.
Not only can the activities of individual enzyme catalysts be determined independently
of other enzymes but the effects of inhibitors can also be analyzed. This approach
enables high throughput analysis on a microchip.
A centrifugal microchip that utilizes a CD player-like apparatus has also been
described (Lai et al., 2004). This microreactor does not require the usual pumping
system, rather centrifugal and capillary forces are used. This method has been applied
to an enzyme-linked immunosorbent assay (ELISA), with each step of the ELISA
process carried out by controlling the rotation speed. This method may be useful
for the development of analytical microbioreaction systems for multiple analyses of
single samples.
Numerical simulation is a strong tool to understand fluid flow and chemical
reactions within microchannels. This technique has also been applied to understand
the mechanism of enzyme reactions in a microchannel (Sotowa et al., 2008). Such
investigations will disclose the mechanism of improved efficiency of enzyme reactions
in a microfluidic format.

Fundamental techniques for enzyme immobilized microreactor

ENZYME-IMMOBILIZATION WITHIN MICROCHANNELS

In the development of enzyme processes, the use of immobilized enzymes is

preferable. Several methods have been available to immobilize enzymes on supports
in conventional reaction apparatus, and these techniques have also been applied to
immobilize enzyme within a microspace (Table 1).
In batchwise reactors, immobilization of enzymes on beads or monoliths has
been used for the separation and recycling of enzymes. This approach has also been
applied to microreaction systems. Microreactors with enzyme immobilized on glass
beads have been prepared by simply filling the reaction chamber with enzyme-
immobilized particles. Such a device was used for the determination of Xanthine
using chemiluminescent detection (Richiter et al., 2002). Crooks and co-workers have
developed advanced analytical microreactors using enzyme-immobilized microbead-
mixing (Figure 1a) (Seong and Crooks 2002), and have efficiently performed multistep
enzyme reactions using glucose oxidase- and horseradish peroxidase-immobilized
polystyrene. In addition, the immobilization of enzyme on Ni-NTA-agarose bead has
also been reported. This immobilized enzyme is less denaturated because binding of
the enzyme is achieved using a His-tag. This method has been applied to immobilize
bacterial P450 (Srinivasan et al., 2004). A similar approach was applied to immobilize
Table 1. Typical techniques for enzyme immobilized microchannel reactor preparation.
410

Technique Media Immobilization method Immobilized enzyme Advantage and disadvantage Ref.

Particle Glass Cross-linking • Xantin oxidase • Ease in preparation Richiter et al.

entrapment (3-aminopropylsilane/ • Horseradish peroxidase • Enable multistep reaction 2002
glutaraldehyde) • Limited number of enzymes are applicable due
to denaturation
• Pressure gain
Polystylene Biotin-Avidin (Avidin- • Horseradish peroxidase • Ease in preparation Seong and
M. Miyazaki et al.

coated beads were used) • Glucose oxidase • Enables multistep reaction Crooks 2002
• Biotin-label is required
• Pressure gain
Agarose Complex formation (Ni-NTA • Bacterial P450 • Ease in preparation Srinivasan et al.
and His-tag) • Applicable for engineered enzymes 2004
• Higher pressure by increasing flow rate and
particles may be crushed
Polystylene Complex formation (Ni-NTA • Benzaldehyde liase • Ease in preparation Drager et al. 2007
and His-tag) • p-Nitrobenzyl esterase • Applicable for engineered enzymes
• Higher pressure by increasing flow rate and
particles may be crushed
Magnetic bead Cross-linking • Glucose oxidase • Preparation is easy Nomura et al.
(3-aminopropylsilane/ • Trypsin • Enzyme can be immobilized on any place 2004
glutaraldehyde) by placing a magnet Li et al. 2007
• Amount of enzyme particle is limited because
of plugging
Polymer monolith Entrapment(2-vinyl-4,4- • Trypsin • Stablization of enzyme structure and activity Sakai-Kato et al
dimethylazlactone, • Requirement of skill in preparation 2004
ethylenedimethacrylate, • Denaturation during entrapment process
2-hydroxyethyl methacrylate,
acrylamide)
Table 1. Contd.
Technique Media Immobilization method Immobilized enzyme Advantage and disadvantage Ref.
Silica monolith Entrapment within porous • Trypsin • Stablization of enzyme structure and activity Sakai-Katoet al.
silica • Protease P • Compatibility in organic solvent 2003
• Requirement of skill in preparation Kawakami et al
• Denaturation possible during entrapment process 2005, 2007
Aluminium oxide Cross-linking • Horseradish peroxidase • Large surface area due to porous nature Heule et al. 2003
(3-aminopropylsilane/ • Applicable for heterogeneous reactions
glutaraldehyde) • Complicated preparation
• Not applicable for large-scale processing

Surface SiO2 surface Physical adsorption of • Alkakine phosphatase • Ease in preparation Gleason and
modification biotinylated poly-lysine • Requirement for avidin- conjugation Carbeck 2004
/biotin-avidin • Possible occurrence of detachment
PDMS Physical adsorption of lipid • Alkakine phosphatase • Enable immobilization of enzyme on Mao et al. 2002
(O2 Plasma treated) bilayer/biotin-avidin plastic surface
• Possible occurrence of detachment
• Expensive reagents
• Requirement for avidin- conjugation
PDMS Physical adsorption of • Alkaline phosphatase • Enable partial modification of microchannel Holden et al 2004
fibrinogen/Photochemmical • Special equipment is required
reaction of Fluorescein- biotin
Slilicon Cross-linking • Trypsin • Simple operation Eckstron et al.
(3-aminopropylsilane/ • Difficulty in channel preparation 2000
glutaraldehyde) • Poor reproducibility
Fused silica Cross-linking • Cucumisin • Simple operation Miyazaki et al
(Sol-gel modified) (3-aminopropylsilane/ • Lipase • Immobilize ~10 times more enzymes than single 2003, 2004,
succinate) • L-Lactic dehydrogenase layer immobilization and therefore, performs 2005
Enzymatic processing in microfluidic reactors

with higher reaction efficiency Kaneno et al

• Several chemistry is available (amide, disulfide, 2003
His-tag)
411
Table 1. Contd.
412

Technique Media Immobilization method Immobilized enzyme Advantage and disadvantage Ref.
• Need several steps for immobilization
• Reproducibility strongly affected by characteristics
of silica surface

PMMA Cross-linking • Trypsin • Stabilize enzyme under denaturation condition Qu et al 2004
[modified with butyl (Si-O bond between • Complicated preparation method
methacrylate/γ- methyl- modified surface and
acryloxy) propyltrimeth- silica monolyth)
M. Miyazaki et al.

oxysilicane]
PDMS Cross-linking • Trypsin • Stabilizes enzyme under denaturation condition Wu et al 2004
(O2 Plasma treated) (Si-O-Ti or Si-O-Al bond • Complicated preparation method
between titania or alumina
monolyth)
Fused silica Cross-linking between • Lipase • Much larger surface area (1.5 times greater than Nakamura et al
physically- immobilized sol-gel modified surface) and higher efficiency 2004
Silica particle • Complicated preparation method
(3-aminopropylsilane/ • Unstable withed physical force (bending etc.)
succinate)

Silicon rubber Cross-linking • Thermophilic β- • Reaction can be performed at 80oC Thomsen et al.
(3-aminopropyltrieth- glycosidase • Complicated preparation method 2007
oxysilane and glutaraldehyde) • Reaction is slow because not much enzyme
can be immobilized
Photopatterning onto Cross-linking by photo- • Horseradish peroxidase • Reduced non-specific absorption Logan et al. 2007
PEG-grafted surface patterned vinylazlactone • Glucose oxidase • Sequentially multistep reaction could be achieved
• Requires special equipment

PDMS Entrapment within hydrogel • Alkaline phosphatase • Quite fast reaction (90% conversion at Koh and Psiko
formed on surface • Urease 10 min reaction) 2005
• Immobilization of multiple enzyme
• Complicated preparation method
• Not applicable for higher flow rate
Technique Media Immobilization method Immobilized enzyme Advantage and disadvantage Ref.
Membrane PDMS/Glass Place PVDF membrane that • Trypsin • Easy preparation Gao et al. 2001
adsorbs enzymes • Less efficiency
• Possibility of leakage at higher flow rate
Glass Covalent cross-linking with • Horseradish peroxidase • Integration of membrane permeation and Hisamoto et al.
Nylon membrane formed at enzyme reaction 2003
liquid-liquid interface • Preparation of multiple membrane
(glutaraldehyde) • Complicated preparation method
• Unstable membrane at higher flow rate
PTFE Enzyme-embedded • α-Chimotrypsin • Easy preparation Honda et al.
membrane formation using • Trypsin • Durable (>40days) 2005, 2006, 2007
glutaraldehyde/ • α-Aminoacylase • Applicable in organic solvents
paraformaldehyde • Other various enzymes • Almost all enzymes can be immobilized by
adding poly-Lys

Enzymatic processing in microfluidic reactors
413
414 M. Miyazaki et al.

Figure 1. Images of immobilization technique for micro enzyme reactor The enzyme can be easily
immobilized by trapping enzyme-immobilized polystylene beads within the microchannel (a). Modified
surfaces are also useful for enzyme immobilization. Modified surface obtained by sol-gel technique (b),
functionalized microstructure fabricated from silicone rubber (c), nanoparticle arrangement (d), and
hydrogel formation (e). Membrane formed within the microchannel can also be used as support for enzyme
immobilization. Nylon membrane formed at liquid-liquid interface (f) or membrane of cross-linking enzyme
aggregate formed at microchannel surface (g) was used for immobilization. These images were reproduced
with permissions from (a) Seong and Crooks (2002), (b) Miyazaki et al. (2003), (c) Thomsen et al. (2007),
(d) Wang et al. (2002), (e) Koh and Pishko (2005), (f) Hisamoto et al. (2003), (g) Honda et al. (2005).
 Enzymatic processing in microfluidic reactors 415

enzymes onto Merrifield resin (Drager et al., 2007). A tyrosine-based Ni-NTA

linker was created on the surface of the resin to immobilize His-tagged enzymes.
This matrix was loaded into a microstructured channel of the PASSflowTM system.
Synthesis of (R)-benzoin, (R)-2-hydroxy -1-phenylpropan-1-one, and 6-O-acetyl-D-
glucal were performed using this system. Magnetic beads were also used for enzyme
immobilization within the microchannel. Glucose oxidase was immobilized within a
Teflon tube with the aid of a magnet (Nomura et al., 2004). The enzyme-immobilized
magnet particles were stable and active for more than eight months. This approach was
also applied for the preparation of protease-immobilized microreactor for proteomic
analysis (Li et al., 2007).
Monolitic microreactors were prepared using several methods. A trypsin-
immobilized microreactor was prepared by molding a porous polymer monolith,
prepared from 2-vinyl-4,4-dimethylazlactone, ethylene dimethacrylate, and acrylamide
or 2-hydroxyethyl methacrylate, with an enzyme, in a microchannel (Sakai-Kato
et al., 2004). This microreactor was used for mapping protein digested fragments.
Preparation of a microreactor by filling a silica monolith made from tetraethoxysilane
with an enzyme and entrapping it within a microchannel has also been developed.
Trypsin-encapsulated monolith was fabricated in situ on a PMMA microchip to
produce an integrated bioreactor that can perform enzymatic digestion, electrophoretic
separation and detection in one chip (Sakai-Kato et al., 2003). Another example is
a protease-P including monolith prepared from a mixture of tetramethoxysilane and
methyltrimethoxysilane (1:4), and that has been used to fill in a PEEK [poly(ether ether
ketone)] microcapillary to produce a microreaction system (Kawakami et al., 2005).
Aluminum oxide powder can be used as a solid support. Horseradish peroxidase was
immobilized on aluminium oxide with 3-aminopropylsilane, and then placed within
the microdevice (Heule et al., 2003). This method takes advantage of the porous
nature of ceramic microstrut. Overall, preparation of the immobilized enzyme with
powdered material or a monolith is significantly easier; however it is unfavorable in
large scale processing because of increasing pressure.

IMMOBILIZATION OF ENZYME ON MICROCHANNEL SURFACE

Methods for enzyme immobilization on the microchannel surface have also been
developed because they can take advantage of the larger surface area of microreaction
systems without gaining pressure. Physical immobilization is an easy way to immobilize
molecules. In microchannel systems, a biotin-avidin system has been mainly used to
immobilize enzymes. The biotinylated polylysine was physically immobilized onto
a glass surface to immobilize streptavidin-conjugated alkakine phosphatase (Gleason
and Carbeck 2004). This microreactor was used for rapid determination of enzyme
kinetics. A biotinylated lipid bilayer (Mao et al., 2002) and partial biotinylation by
photo patterning on fibrinogen (Holden et al., 2004) were also used for immobilization.
However, these methods are not suitable for long-term use because of their instability.
Also, applications are limited to streptavidin-conjugated enzymes.
The introduction of a functional group on the microchannel surface was used
for covalent cross-linking. A trypsin-immobilized microreactor was prepared by
modification with 3-aminopropylsilane and glutaraldehyde using the classical method
(Eckstrom et al., 2000). Although this immobilization method is easy, fabrication of a
416 M. Miyazaki et al.

complexed microstructure is required to obtain high performance. Our group developed

a modified sol-gel technique to form nanostructures on a silica microchannel surface
(Figure 1b) (Miyazaki et al., 2003). This method modifies the microchannel surface
with polymerized copolymer of 3-aminopropylsilane/ methylsilane. Using this method,
an increased surface area has been obtained. Enzymes can be immobilized on these
nanostructures by covalent cross-linking through amide-bond formation, disulfide or
His-tag, by modifying succinate spacer, at least 10 times more compared with single layer
immobilization (Kaneno et al., 2003; Miyazaki et al., 2004; Miyazaki et al., 2005). A
microreactor with immobilized cucumisin on the nanostructured surface could process
substrate 15 times faster than the batchwise reaction (Miyazaki et al., 2004).
Similar surface modification methods employing sol-gel technique have also been
developed (Qu et al., 2004). A PMMA surface was modified with a copolymer of
butyl methacrylate/γ- (methylacryloxy)propyltrimethoxysilicane and silica-sol-gel
to immobilize enzymes. Using this method, a trypsin-immobilized microreactor was
developed. In addition, a trypsin-encapsulated titania and alumina gel matrix was
immobilized through the SiOH group formed on a PDMS surface by plasma oxidation
(Wu et al., 2004). Using this device, the digestion time was significantly shortened
(ca. 2s) and the application for high-throughput protein identification was realized.
Alternatively, silicone rubber material was used for the preparation of functional
nanostructure on the microchannel surface (Figure 1c: Thomsen et al., 2007). The
structure was prepared by micromould fabrication using vinyl-group containing PDMS
and silicic acid, and enzyme immobilization by cross-linking with glutalaldehyde.
Using this procedure, a microstructured enzyme reactor with immobilized thermophilic
β-glycosidasecapable of performing hydrolysis at 80oC was created.
A particle-arrangement technique was also applied to enzyme immobilization.
Silica nanoparticles were immobilized onto the surface using a slow evaporation
of particle suspension filled-in microchannel (Figure 1d) (Wang et al., 2002). The
microchannel obtained was subjected to treatment with 3-aminopropyltriethoxysilane,
and immobilization of enzyme was achieved by covalent cross-linking through
amino group. Although physical stability needs to be improved, lipase-immobilized
microreactor prepared by this method showed 1.5 times faster kinetics than that
of microreactor obtained by sol-gel surface modification (Nakamura et al., 2004).
This result showed good correlation with the surface area; particle arrangement has
approximately 1.5 times larger surface area and could immobilize more enzymes.
Photochemistry was applied to enable selective immobilization of enzymes on
the microchannel surface (Logan et al., 2007). In the procedure, vinyl azlaction was
photo-grafted onto a PEG-coated polymer surface as a reactive monomer and the
enzymes were immobilized through their amino group. This approach was applied for
the immobilization of horseradish peroxidase. Another approach for efficient enzyme
immobilization is polymer coating. Poly(ethylene glycol)based-hydrogels which
incorporate alkaline phosphatase was prepared within a microchannel by exposure
to UV light (Figure 1e) (Koh and Pishko 2005). This method was also applied to
immobilize urease and different enzymes on microchannel surfaces.

MEMBRANE-FORMATION

Enzymes can be immobilized on a membrane within the microchannel. A porous

 Enzymatic processing in microfluidic reactors 417

poly(vinylidene fluoride) membrane embedded within a microchannel can be used for

enzyme immobilization. Preparation of a miniaturized membrane reactor by absorption
of enzymes onto the membrane has been reported (Gao et al., 2001).
Hisamoto and colleagues have demonstrated that a nylon-membrane formation at
the interface of two solutions occurred in a microchannel (Figure 1f). Peroxidase was
immobilized on this membrane which was used as a chemicofunctional membrane
(Hisamoto et al., 2003); however, immobilization of the membrane is technically
difficult, and application of this method is limited because the nylon-membrane is
unstable in organic solvents.
We have developed a technique that forms an enzyme-immobilizing membrane on
the microchannel surface (Honda 2005). This is a modification of CLEA (cross-linked
enzyme aggregate) formation, which is used in batchwise organic synthesis (Cao et
al., 2006). Simple loading of the enzyme solution and a mixture of glutaraldehyde
and paraformaldehyde into the microchannel forms a CLEA membrane on the
microchannel wall (Figure 1g). The resulting microreactor can be used for prolonged
periods (>40 days), and shows excellent stability against organic solvent. Taking these
advantages into account, this method is considered ideal for the development of an
enzymatic reactor tailored for specific applications. However, this method requires an
amino group for immobilization, and application of acidic enzymes with few amino
group on their surface is difficult. The application of the approach developed in our
laboratory was expanded by adding poly-Lys to aid in membrane formation of acidic
proteins (Honda et al., 2006). By this method, almost all enzymes, including highly
acidic proteins, can form cross-linked aggregates. We applied this technique to the
preparation of an enzyme microreactor, and demonstrated immobilization of several
acidic enzymes by this method (Honda et al., 2006). Our results have indicated that
almost all enzymes can be immobilized onto the microchannel surface, and that our
approach is a robust way of enzyme-immobilized microreactor development.

Advanced applications of micro enzyme reactor for processing

SOLUTION-PHASE ENZYME REACTIONS

Numerous analytical micro-enzyme reactors that take advantage of the reduction of

time and the minimal amount of reagents used in microchannel systems (Krenkova
et al., 2004) have been developed. However, there have been only a few reports on
continuous-flow enzymatic microreaction processes (Table 2).
Although the solution-phase reaction is not a favorable process due to the large
volume of enzyme required, several important achievements have been reported.
Enzymatic oligosaccaride synthesis was performed using β-galactosidase in a
continuous-flow microreactor (Kanno et al., 2002a,b). The reaction was performed
by separate loading of the enzyme in phosphate buffer solution and the substrate
solution in acetonitrile into inlets, and was terminated by heating the recovered
solution. The reaction in the microchannel was about 5 times faster than that in the
batch reaction.
A biphasic continuous-flow microreaction has also been devised. Goto and co-
workers have performed dehalogenation reactions in a chip-type glass microreactor
using laccase by separately loading an aqueous solution of enzyme and substrate
Table 2. Enzymatic processing performed in micro enzyme reactor.
418

Reaction Technique Enzyme Results Ref.

HO Solution-phase • ß-Glucosidase • 5 times better yield was Kanno et al.
HH
N O
HO O continuous-flow obtained than that of 2002a, 2002b
NO2
HO H O H CH
OH H 3 batchwise reaction
H
OHHO H OH H H OH • Isomers were not isolated
H HO H
O OH
H
O
HO H OH OHHO
M. Miyazaki et al.

H H H CH3
H O HO H
NO2 O HN O
HO H OH H OH
H H H
H HO
H H O H
HO O OH
O
HO H NH
H H
OHHO
O CH3 H
O HO
H H
O
HO H OH
H OH
H H
H
O H O
NH
H3C H
OH

Biphasic solution- • Laccase • Degradation of p-chlorophenol Maruyana et al.

Cl O
phase continuous occurred mainly at the aqueous- 2003
flow reaction organic interface in the
microchannel
• Diffusion of the substrate is
OH O
the rate-limiting step in the
enzymatic degradation
Plug-flow • Hydroxynitrite • Asymmetric synthesis was Koch et al, 2008
O 1eq. HCN, Enzyme OH biphasic system lyase achieved
• Product can be separated from
R H MTBE/ buffer ph5.0 R
N enzyme solution and obtained as
a MTBE solution.
Table 2. Contd.

Reaction Technique Enzyme Results Ref.

O Solution-phase • Formate • Regenerate coenzymes within Yoon et al. 2005
OH
H3C reaction using dehydrogenase single reactor
+
FAD NAD O
electrochemical • Lactate • Regeneration of NADH was as
microreactor dehydrogenase high as 31%

e- FADH2 NADH H3C OH

OH
H
O

O Ni-NTA • PikC • >90% conversion was obtained Srinivasan et al.,

CH3
CH3
HO agarose bead hydroxylase at 70nm/min 2004
CH3
O H3C O
CH3 CH3
immobilization (Bacterial
CH3 O N
O P450)
H3C CH3 CH3 OH CH3
O
CH3
O N
O
CH3 OH CH3
O
CH3
CH3
H3C CH3
O
HO CH3
O N
O
CH3 OH CH3

Benzoin reaction Ni-NTA • Benzaldehyde • >80% yields were obtained Drager et al.
O agarose bead liase 2007
H H R
+ R immobilization • p-Nitrobenzyl
O O OH
esterase
Regioselective hydrolysis
Enzymatic processing in microfluidic reactors

O O O
OAc OAc HO
+
OAc HO HO
OAc OH OH
419
Table 2. Contd.
420

Reaction Technique Enzyme Results Ref.

Entrapment • Zinc • Used combinatorial synthesis of Luckarift et al.
NO2 NHOH NH2 of silica- • Hydroxyami- 2-aminophenoxyazin-3-one 2007
OH N NH2
immobilized nobenzene
O O enzymes within mutase
microchannel • Peroxidase
M. Miyazaki et al.

Surface • L-Lactic • Crude enzyme can be used for Miyazaki et al.

modification by dehydrogenase immobilization 2005
O OH sol-gel technique/ • Reversible immobilization was
OH OH Ni-NTA
H3C H3C achieved by EDTA treatment
O O immobilization • Reaction was completed within
15 min

Surface • Lipase • 1.5 time better yield was Kaneno et al.

modification of obtained compared with 2003
silica capillary by batchwise reaction
O sol-gel technique/
immobilized
H3C O O O HO O O
through amide
bond formation
using succinate
linker
HO O OH Entrapment • Novozym-435 • Much less of the reactant was Garcia et al. 2000
H3C OH
OH OH
O OH
of Novozym- required compared with the
O OH O 435TM within batchwise test
O OH
H3C microchannel
Table 2. Contd.

Reaction Technique Enzyme Results Ref.

HO O
Silica monolith • Protease P • Conversion within microreactor Kawakami et al.
entrapped within was higher than that of the 2005
H3C O O
microchannels batchwise reaction at higher
O CH3
O flow rates
O

HO O H3C O O
Silica monolith • Lipase • Optical resolution of products Kawakami et al.
entrapped within was achieved by connecting 2007
O
microchannels commercially available chiral
O CH3
H3C O O column
O
O

Ar Membrane • α-Aminoacylase • Optical resolution of D/L-amino Honda et al. 2007

O
OH Organic phase formation with acids were achieved by connect-
Ar H3C N
O H
O paraformaldehyde, ing to micro solvent extractor
OH
H3C N
H Ar
glutaraldehyde,
O

OH Aqueous phase
and poly-Lys
H2N
O

Enzymatic processing in microfluidic reactors
421
422 M. Miyazaki et al.

solution in organic solvent (Maruyama et al., 2003). These researchers performed a

detailed kinetic analysis and concluded that the reaction kinetics of a biphasic stream
in a microchannel depends on the diffusion of the substrate into the aqueous phase.
A more complicated continuous-flow type enzyme microreaction system was
also developed. Regeneration of coenzyme is the most difficult point in an enzyme-
catalyzed process. Regeneration of NADH was performed in a Y-shape microreactor,
which possesses an electrode within the microchannel (Yoon et al., 2005). Another
example has been reported by Koch et al. (2008). They used a segment-flow system
of methyl-tert-butyl ether and aqueous buffer for asymmetric synthesis of (S)-
cyanohydrins catalyzed by hydroxynitrile lyase. In their system, clude cell lysate
could be used. Although these experiments are primitive, these results demonstrate
that a continuous-flow microreactor is one of the more promising devices for the
development of efficient enzyme reaction systems.

ENZYME-IMMOBILIZED MICROREACTOR FOR PROCESSING

Applications of enzyme-immobilized microreactors for processing have also been

presented including hydroxylation of macrolide in a microreactor (Srinivasan et
al., 2004). PikC hydroxylase was immobilized on Ni-NTA agarose beads, and
then filled into the microchannel. This microreactor was used for hydroxylation to
produce methymycin and neomethylmycin, and over 90% conversion was achieved
at a flow rate of 70nl/min. Such high efficiency might have resulted from shorter
residence times, which is preferable for enzymes with inherent stability. A similar
immobilization technique was applied for the synthesis of (R)-benzoin, (R)-2-hydroxy-
1-phenylpropan-1-one, and 6-O-acetyl-D-glucal (Drager et al. 2007). His-tagged
protein was directly immobilized within the microstructured PASSflow reaction system
through tyrosine-based Ni-NTA system.
The application of enzyme-immobilized microreactor for multistep synthesis
has also been demonstrated (Luckarift et al., 2007). Three separated microfluidic
devices, which possess metallic zinc, silica-immobilized hydroxyaminobenzene
mutase, and silica-immobilized peroxidase within a microchannel, were prepared
and connected sequentially. These devices were used for combinatorial synthesis of
2-aminophenoxyazin-3-one. These results open the door for the application of micro
bioreactors for enzymatic synthesis of bioactive natural products.
Esterification and hydrolysis reactions are important processes in industry that have
also been performed in a microchannel system. Lipase-immobilized microreactors
have been prepared using a ceramic microreactor and glass microcapillary (Kaneno et
al., 2003), wherein hydrolysis of the ester was conducted. Both microreactors showed
1.5 times better yield than the batchwise reaction using the same volume/enzyme ratios.
This could have resulted from an increase in contact due to the larger surface area of
microchannel systems. A microreaction using immobilized Novozym-435TM was also
reported, where esterification of diglycerol with lauric acid was performed (Garcia et
al., 2000). A monolytic microreactor tethering protease P was applied for bioconversion
process. Transesterification of (S)-(-)-glycidol and vinyl n-butyrate was performed
using this microreaction device (Kawakami et al., 2005) but the conversion depended
on the amount of immobilized enzyme. Similary, they separated a racemic product
 Enzymatic processing in microfluidic reactors 423

which was obtained by reaction in a lipase-entrapped microreactor, by connecting

chiral column sequentially to the microreactor (Kawakami et al., 2007).
We have developed a novel integrated microreaction system which combined an enzyme
microreactor with a solvent extractor. The enzyme-immobilized microreactor was prepared
by membrane-formation technique using α-aminoacylase with poly-Lys (Honda et al.,
2006). This microreactor was connected with a microextractor which has a partially modified
microchannel (Yamaguchi et al., 2005). Using this microreaction system, optical resolution
of D/L-phenylalanine analogs was performed. The D-phenylalanine analogs were obtained
efficiently with high optical purity (Honda et al., 2007).
So far, few enzymes have been applied for microreaction process development.

Concluding remarks

Microchannel devices can be useful in imitating various biological reaction apparatus,

such as the cellular surface and vascular system, by providing the advantages of limited
space and laminar flow compared with the conventional reaction apparatus. The quest
for microreaction technologies will lead to better process intensification and efficient
analytical methods. Increasingly, new findings are being achieved in microfluidics.
Further investigation on microfluidics could provide novel mechanisms not observed
in conventional systems, and better understanding of fluidics in microchannels might
enable new reaction pathways not possible with the conventional systems.
The strong advantages offered by microreaction devices are useful, particularly in the
development of microreaction systems for commercial purposes. Once a microreactor
has been optimized, it can be easily introduced into an industrial-scale plant. Parallel
scale-out enables extension of reaction conditions optimized in a single reactor, and
eliminates scale-up problems arising from conventional processes. Parallel operation
of the same microreaction provides high throughput operation of different reagents at a
single operation and serves as an excellent tool for combinatorial processing. Although
several problems, such as connection, parallel control of fluid and reaction conditions,
and monitoring, are common challenges, the benefits offered by microreaction
technology accelerate the development of enzyme reaction devices.
As described here, few enzymes have been applied to microreaction process
development, and not so many patents describing the construction of micro enzyme
reactors have been thusfar published. This is a clear indication that the field is still in
its initial stage. Efforts directed to the development, optimization and application of
micro enzyme reactors will open a new era for biochemical processing.

Acknowledgments

We thank Professors Shigeharu Morooka (Fukuoka University), (Fukuoka Women’s

University), Masayuki Fujii (Kinki University), Kazunari Arima (Kagoshima
University) and Kenichi Kanno (Kinki University) for their support. The technical
assistance of Jun Kaneno (NS Materials Co.) and other colleagues in the laboratory
are also acknowledged. Part of this work was supported by grants-in-aid for scientific
research (B) (20310074) from Japan Society for the Promotion of Science.
424 M. Miyazaki et al.

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