Enzymatic microreactors in chemical analysis and kinetic studies

Enzymatic microreactors in chemical analysis and kinetic studies
Abstract
Pawel L. Urban a , David M. Goodall a, *, Neil C. Bruce b
a Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK
b CNAP, Department of Biology, University of York, Heslington, York, YO10 5YW, UK
Received 30 May 2005; accepted 3 June 2005
Available online 1 August 2005
The fields of application of microreactors are becoming wider every year. A considerable number of papers have been published
recently reporting successful application of enzymatic microreactors in chemistry and biochemistry. Most are devices with enzymes
immobilized on beads or walls of microfluidic channels, whilst some use dissolved enzymes to run a reaction in the microfluidic
system. Apart from model systems, mostly with glucose oxidase, horseradish peroxidase and alkaline phosphatase, the principal
fields of application of microreactors are tryptic digestion of proteins and polymerase chain reaction in automated analyses of
proteomic and genetic material, respectively. Enzymatic microreactors also facilitate characterization of enzyme activity as a
function of substrate concentration, and enable fast screening of new biocatalysts and their substrates. They may constitute key
parts of lab-on-a-chip and ATAS, assisting the analysis of biomolecules. This review provides systematic coverage of examples of
reports on enzymatic microreactors published recently, as well as relevant older papers.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Characterization of proteins and DNA; Enzyme immobilization; Lab-on-a-chip; Microfluidic systems; Microreactors
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2. Applications of enzymatic microreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.1. Analysis of chemical species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.1.1. Homogeneous and heterogeneous biocatalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.1.2. Analysis of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.1.3. Analysis of nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.1.4. Model enzymatic systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.2. Other applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.2.1. Kinetic studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.2.2. Imaging of biotransformations in microreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3. Conclusions and future trends. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

1. Introduction
Microreactors are usually defined as miniaturized
reaction systems fabricated by using, at least partially,
methods of microtechnology and precision engineering
(Ehrfeld et al., 2000). The term bmicroreactorQ is the
proposed name for a wide range of devices having
small dimensions, and a further division according to
size into nano, micro and minireactors is hardly ever
used (Ehrfeld et al., 2000). Most of the currently constructed
microreaction devices take advantage of microfluidics
and nanofluidics, which enables use of micro
and nanolitre volumes of reactive species and ensures
high efficiency as well as repeatability of biocatalytic
processes. Microreactors find applications in organic
synthesis (Haswell and Watts, 2003; Hessel et al.,
2004). An example of an application in biotechnology
is the fast multi-step synthesis of peptides (Watts et al.,
2001). The main benefits of application of microreactors
in industry are: faster transfer of results of development
work into production, earlier start of production
at lower costs, easier scale-up of production capacity,
smaller plant size, lower costs for transportation, materials
and energy, and more flexible response to market
demands (Ehrfeld et al., 2000).
Though the majority of papers describe microreactors
as components of microfluidic devices, this term is
also used in context of self-organised systems such as
reverse micelles (Madamwar et al., 1988; Chopineau et
al., 1998; Carvalho and Cabral, 2000), liposomes (Oberholzer
et al., 1999; Walde and Ichikawa, 2001) and
microemulsions (Garti et al., 1997; Garti, 2003). Selforganised
systems will not be discussed in this review.
Analytical systems which comprise microreactors are
expected to be characterized by outstanding repeatability
and reproducibility, due to replacing batch iterative steps
and discrete sample treatment by flow injection systems.
The possibility of performing similar analyses in parallel
is an attractive feature for screening and routine use.
Microreactors have been integrated into automated analytical
systems (Pfohl et al., 2003), and as well as providing
benefits from system automation this also
eliminates errors associated with manual protocols. Further
advantages of the use of microreactors in analytical
chemistry are that they can be coupled with numerous
detection techniques (Schwarz and Hauser, 2001; Verpoorte,
2003a,b), and that pretreatment of the samples
can be carried out on the chip (de Mello and Beard, 2003;
Chiesl et al., 2005). Methods of injection of the fluids
into microchannels, and connecting and interfacing
microreactors with other system components, are also
being improved. This should help eliminate any remain-
ing obstacles to more widespread uptake of the technology
(Fang, 2004). Miniaturized analytical assays are
useful in many branches of biotechnology (Guijt-van
Duijn et al., 2003). The influence of nanotechnology in
the development of biosensors has been reviewed by
Jianrong et al. (2004). Whilst recently published books
cover industrial applications of microreactors (Ehrfeld et
al., 2000; Hessel et al., 2004, 2005a,b), analytical applications
are of increasing importance and are therefore
also surveyed in the present review.
Enzymatic microreactors have been developed in
order to facilitate routine work in biochemical analysis,
and also have applications in biocatalysis. A low expenditure
of the enzyme is often a result of its immobilization.
However, the range of immobilized enzymes
available with satisfactory characteristics is still limited
(Buchholz et al., 2005), which inevitably decreases the
number of potential applications. The following immobilized
enzymes are used on an industrial scale: glucose
isomerase, sucrose mutase, h-galactosidase, penicillin
acylase, d-amino acid oxidase, glutaryl amidase, thermolysin,
nitrilase, aminoacylase and hydantoinases
(Buchholz et al., 2005).
Enzymatic microreactors have been used for analytical
applications as components of integrated systems,
often termed lab-on-a-chip or in micro total analysis
systems (ATAS) (Vilkner et al., 2004). Although the
first enzymatic microreactors were constructed in the
1970s and 1980s, the growth in their practical applications
dates to the late 1990s. No examples of enzymatic
microreactors were included in the first comprehensive
book on microreactors published in 2000 (Ehrfeld et al.,
2000).
It is helpful to divide the analytical applications of
enzymatic microreactors into two classes. Firstly,
those which use biocatalysis in order to transform
an analyte difficult to measure into an easily measurable
form. Secondly, microreactors designed for
screening of substrates, enzymes and examine their
kinetic characteristics. The first category is exemplified
by the large number of microsystems designed
for digestion of proteins to convert them to morereadily
measured peptides. Another example is oxidation
of glucose by glucose oxidase followed by
measuring chemiluminescence of luminol oxidised
by hydrogen peroxide formed in the primary reaction
(L’Hostis et al., 2000). The second category is exemplified
by work presented by Seong et al. (2003) to
quantitatively measure enzyme kinetics in a continuous-flow
microfluidic system.
The aim of this review is to summarize recent
work in the field of enzymatic microreactors, which

constitutes a new branch of microtechnology. Objectives
are to highlight new arising trends in the
development of enzymatic microreactors, to show
their present applications in applied analytical chemistry
and biochemical studies, and to consider possible
implications of enzymatic microreactors in
biotechnology.
2. Applications of enzymatic microreactors
The achievements in chemical and biochemical
microreaction systems before 2000 have been highlighted
by Haswell and Skelton (2000). Developments
in immobilized microfluidic enzymatic reactors have
been discussed by Krenková and Foret (2004). Girelli
and Mattei (2005) have recently reviewed the applications
of immobilized enzyme reactors in high performance
liquid chromatography: most of the constructed
reactors used enzymes bound covalently to the support,
and the functional groups involved in the binding were
amino, epoxyl, carboxyl, diol and phenolic. Examples
were given of enzyme-catalysed reactions carried out
before or after the column separation, as well as in the
column.
2.1. Analysis of chemical species
This section commences with coverage of the types
of microreactors used, with the classification into homogeneous
and heterogeneous biocatalysis. All of the
examples of successful application of enzymatic microreactors
with immobilized enzymes presented in this
review can be classified into the following groups: (i)
analysis of proteins, (ii) analysis of nucleic acids, and
(iii) model enzymatic systems. Separate subsections
describe each in turn.
2.1.1. Homogeneous and heterogeneous biocatalysis
Microreactors may use an immobilized enzyme, or
its solution may be injected to the reaction zone; the
two approaches encompass heterogeneous and homogenous
biocatalysis, respectively. The majority of published
applications refer to use of immobilised
enzymes. An example of homogeneous biocatalysis
on a microscale is electrophoretically mediated microanalysis
(EMMA), first described by Bao and Regnier
(1992) and later referred to as EMMA (Regnier et al.,
1995), which makes use of different mobility of an
enzyme and its substrate in order to mix the zones of
both and to accomplish bioconversion of the substrate
to the product. The enzyme solution is injected into a
fused silica capillary followed by injection of its sub-
strate, and the capillary is considered as the microreactor
(Avila and Whitesides, 1993; Van Dyck et al.,
2003). In spite of the apparent complexity, such a
configuration is suitable for automation and control of
the time of contact between the catalyst and its substrate.
However, the EMMA method is only feasible in
the case of fast catalytic processes, since contact times
are typically in the range milliseconds to seconds.
EMMA is advantageous because the amounts of enzyme
and substrate used are extremely small (Kanie
and Kanie, 2003).
Apart from enzymes, another chemical species (e.g.
an antibody) may be immobilized in the microchannel
and an enzyme involved in the specific reaction may be
injected to the system (Yakovleva et al., 2002). Further
examples of microsystems without immobilized
enzymes involve conversions catalyzed by alkaline
phosphatase (Liu et al., 2004; Moorthy et al., 2004),
glucose oxidase, catalase, urease (Zhang and Tadigadapa,
2004), glycosidase (Kanno et al., 2002) and laccase
(Maruyama et al., 2003), illustrating a wide range
of enzymes that can be applied in such microsystems.
The mode of operation involving homogeneous catalysis
is relatively easy to achieve, since there is no need
for immobilization of the enzyme. A drawback of using
homogeneous biocatalysis and injecting the enzyme
solution to the microreactor is the difficulty of enzyme
recycling, as well as the necessity of its continuous
dosage. Nevertheless, the use of microfluidic components
allows the reactions to be achieved in nanolitre
volumes without excessive expenditure of biocatalytic
species.
Use of microreactors with enzymes immobilized
either on packed beads or on the inner wall of the
channel does not require continuous supply of the
biocatalyst within the run. The substrates are normally
moved through the channel by application of pressure.
An alternative is by use of electroosmotic flow (Haswell
and Skelton, 2000), which is potentially attractive
with microchannels packed with small particles which
would provide high backpressure. More information on
the solid supports used in microreactors can be found in
the review by Peterson (2005).
2.1.2. Analysis of proteins
The greatest number of recent applications of enzymatic
microreactors refer to protein and peptide mapping
(Table 1), an essential process in the identification
and sequencing of proteins. The most frequently used
enzyme is trypsin, the enzyme catalyzing the process of
protein digestion through hydrolysis of peptide bonds at
a basic residue.

Table 1
Application of enzymatic microreactors in the analysis of proteins
Enzyme Medium Application Reference
Chymotrypsin Silicon Protein identification Ekström et al., 2000
Chymotrypsin,
trypsin, papain
Pepsin Gel on a photopolymerized
porous silica monolith
Magnetic microparticles Specific fragmentation
of high molecular-mass
and heterogeneous
glycoproteins
One example of such a system involves a homemade
microreactor with trypsin immobilized on controlled
pore glass (CPG) beads (Bonneil et al., 2000),
Protein digestion,
peptide separation,
and protein identification
Peptide mapping analysis
of proteins
Korecka et al., 2004
Kato et al., 2004
Protease Fused-silica capillary (metal–ion
chelated adsorption )
Guo et al., 2003
Protease Porous wall of a capillary Protein mapping Guo et al., 2002
Trypsin Controlled pore glass On-line protein digestion,
preconcentration, separation
and detection (in UV)
Bonneil and Waldron, 2000
Trypsin Controlled pore glass Peptide mapping Bonneil et al., 2000
Trypsin Fused-silica capillary Analysis of proteins
and peptides
Licklider et al., 1995
Trypsin Fused-silica capillary Digestion of proteins Licklider and Kuhr, 1998
Trypsin Fused-silica capillary On-column digestions
of small amounts of proteins
Amankwa and Kuhr, 1992
Trypsin Gel beads Digestion of proteins Jin et al., 2003
Trypsin Glycidyl methacrylatemodified
cellulose membrane
Peptide mapping Jiang et al., 2000c
Trypsin Glycidyl methacrylatemodified
cellulose membrane
Peptide mapping Jiang et al., 2000b
Trypsin Glycidyl methacrylate-
On-line frontal analysis
Jiang et al., 2000a
modified cellulose
of enzymatic products
Trypsin Injected Digestion of insulin Gottschlich et al., 2000
Trypsin Micropatterned sol–gel
structures in polydimethylsiloxane
microchannels
Protein patterning Kim et al., 2001
Trypsin Monolith Enzyme digestion for
peptide mapping
Peterson et al., 2003
Trypsin Monolith Analysis of proteins Xie et al., 1999
Trypsin Monolithic capillary column Digestion of picomoles
of proteins
Ye et al., 2004
Trypsin Porous polymer monolith Protein mapping Peterson et al., 2002a
Trypsin Porous polymer monolith Peptide mass mapping Palm and Novotny, 2004
Trypsin Porous polymer monolith in
fused-silica capillary
Peptide mass mapping Peterson et al., 2002b
Trypsin, glucose
Porous silicon High-speed on-line
Bengtsson et al., 2002
oxidase
protein digestion
Trypsin Porozyme Protein mapping
(lactate dehydrogenase)
Samskog et al., 2003
Trypsin PVDF membrane disk Extraction of proteins
from 2D gels and digestion
Cooper and Lee, 2004
Trypsin RP beads Protein mapping Ekström et al., 2002
Trypsin Silica gel microchannels Proteomic research Qu et al., 2004
Trypsin and pepsin
(proteases)
Fused-silica capillary Peptide mapping Licklider and Kuhr, 1994
Trypsin poly(vinylidene fluoride)
Rapid protein digestion,
Jiang and Lee, 2001
in Poly(dimethylsiloxane)
peptide separation, and
channel
protein identification
Fig. 1. The microreactor was prepared by dry-packing
into a fused silica capillary (530 Am i.d.) under sonication.
An HPLC column was used as a reservoir contain-

programme is applied to enable amplification of the
DNA chain. Nagai et al. (2001) produced a microarray
with microchambers of dimensions 80 80 Am and
volume 85 pl. Each chamber could contain as little as
a single molecule prior to DNA amplification. Ke et al.
(2004) designed a silicon-based system with an internal
cavity volume of 7 Al for carrying fast detection of the
DNA sequence characteristic for Mycobacterium tuberculosis,
based on mutation in the 81 bp region of rpoB
gene. In comparison with current methods for diagnosis
of tuberculosis, this provides significant savings in time
and cost of enzymes and reagents.
A few authors have described microdevices for further
analysis of DNA, including its digestion with
restrictases (Washizu et al., 1996; Burns et al., 1998;
Katsura et al., 2004). Uptake of such systems could
improve automation in providing restriction maps for
plasmids, which are routinely required in many research
protocols in molecular biology. Fig. 2 shows the device
constructed by Burns et al., 1998. It incorporates PCR
as well as separation of the product with capillary
electrophoresis. As little as 10 ng Al 1 of DNA in
120 nl drops could be detected. DNA sequencing in
microsystems has been reviewed by Kan et al. (2004),
and Lagally and Mathies (2004) have covered recent
developments in technologies available for genetic
analysis on a microscale.
Chip-based devices for genomics and proteomics,
including PCR, have been discussed by Sanders and
Manz (2000). Schneegaß and Köhler (2001) reviewed
the development of a variety of devices and components
for performing DNA amplification, and gave a
comparison of batch-process thermocyclers with reaction
chambers and flow-through devices for different
purposes. They pointed out the advantages of using
microdevices, not only because of the size reduction
but also for their greater efficiency. The heating and
cooling elements possess small volumes and heat capacities,
which yield high heating and cooling rates,
typically in the range 15–40 K s 1 . Conventional thermocyclers
achieve heating and cooling rates of approximately
2–10 K s 1 , a factor of 4 or more lower than
the microdevices. Kricka and Wilding (2003) have
discussed general aspects of miniaturization trends
concerning PCR. Hashimoto et al. (2003) have described
a ATAS for DNA analysis including amplification,
purification, sequencing and separation, and have
recently highlighted the influence of flow rate on the
kinetics of the PCR reaction under continuous flow
conditions (Hashimoto et al., 2004).
Fig. 2. Schematic of integrated device with two liquid samples and electrophoresis gel for nanolitre analysis of DNA. Reprinted with permission
from Burns et al. (1998). Copyright (1998) AAAS.

2.1.4. Model enzymatic systems
A variety of immobilization techniques and microfluidic
designs have been used to build enzymatic
microreactors designed for use in analysis of chemical
species (Table 2). The most widely-used supports for
immobilization of enzymes are beads of silicon or glass,
although there are numerous particular solutions including
immobilization on the inner walls of microfluidic
channels and fused silica capillaries. The most popular
enzymes for testing immobilization efficiency and
assaying the microreaction process are glucose oxidase
(Murakami et al., 1993; Laurell and Rosengren, 1994;
Laurell et al., 1995; Drott et al., 1997; Folly et al., 1997;
Drott et al., 1999; Kulys, 1999; Niwa et al., 1999;
Bengtsson et al., 2000; L’Hostis et al., 2000; Strike et
al., 2000; Bengtsson et al., 2002; Mao et al., 2002; Park
and Clark, 2002; Wilhelm and Wittstock, 2002; Zhan et
al., 2002; Park et al., 2003; Holden et al., 2004;
Nomura et al., 2004; Xu and Fang, 2004), horseradish
peroxidase (Mao et al., 2002; Park and Clark, 2002;
Wilhelm and Wittstock, 2002; Zhan et al., 2002; Heule
et al., 2003; Lv et al., 2003; Seong et al., 2003; Holden
et al., 2004) and alkaline phosphatase (Mao et al., 2002;
Park et al., 2003; Gleason and Carbeck, 2004; Holden
et al., 2004; Koh and Pishko, 2005). These enzymes are
relatively cheap and easily accessible, and their chemical
nature and the reactions catalyzed by them are well
understood. In work by L’Hostis et al. (2000) a microscale
electrochemiluminescence (ECL) detector was
used to monitor the products of conversion of glucose
by glucose oxidase immobilized on glass beads with
luminol as a chelator. It allowed the detection of glucose
within the biologically-relevant range 50–500 AM.
In the near future, it is expected that work using model
enzymes will be augmented by studies with other
enzymes useful for analytical assays.
2.2. Other applications
Apart from applications in analysis of chemical
species, several of the methods presented in the previous
sections are also useful in kinetic characterization
of enzymes. This will be covered in Section 2.2.1.
There have been several attempts to use imaging techniques
to directly visualize within the microchannel the
product formed in the course of reaction. Imaging
techniques applied with enzymatic microreactors are
discussed in Section 2.2.2.
2.2.1. Kinetic studies
Microreactors offer significant advantages for online
monitoring of biocatalysis and characterisation of
kinetics of supported enzymes. Generally, such
enzymes are of better stability than when in free solution
(Cao, 2005). Microreactors enable the key parameters
characterising the kinetics, Km and vmax, tobe
determined for immobilized enzymes. Characterization
of new immobilized enzymes can be facilitated by
using miniaturized systems in continuous flow mode.
Results are obtained using very small quantities of
immobilized enzymes and the methods are readily amenable
to automation of the protocols. Such methods
overcome problems with batch assays for immobilized
enzymes, e.g. the difficulty of mixing of the solid
particles containing supported enzyme with the substrate
solution.
Seong et al. (2003) showed that the Michaelis constant
determined with a microfluidic device with immobilized
horseradish peroxidase was similar to the value
obtained during homogeneous catalysis in batch mode.
An interesting method for determining K m and v max
was presented by Jiang et al. (2000a), who applied online
frontal analysis of peptides originating from the
digestion by trypsin immobilized on glycidyl methacrylate-modified
cellulose. The Lineweaver–Burke diagrams
were easily constructed, based on the effects of
injection of different concentrations and variation of
flow rate of the substrate solution. Bilitewski et al.
(2003) highlighted the application of microfluidic systems
to enzymatic reactions.
In many cases, an enzymatic reaction is very fast and
can reach equilibrium within a single passage of substrate
stream through the microreaction channel. However,
several biotransformations, for example those
catalyzed by lipases, are slower. In these cases, a recirculating
system can be constructed using a loop of
tubing together with the reactor, as in Fig. 3 (Pijanowska
et al., 2001). The substrate solution was
pumped through the system with a peristaltic pump.
Three types of immobilization were tested, and high
performance of the units was demonstrated with either
glass beads or nitrocellulose sheets as enzyme carrier,
while entrapment within alginate gel beads was shown
to give unsatisfactory results. Hydrolysis of the substrate
was measured by change of pH during the initial
phase of the reaction over a 25 min period; the time to
reach the steady-state was estimated at 110 min. Use of
pH measurement to monitor progress of the reaction
was shown to be sensitive, 0.478 pH/mM for tributyrin
(b4 mM). Scaling down the dimensions of the microreactor,
and immobilizing the enzyme (lipase) inside a
fused silica capillary leads to very short times for the
hydrolysis (Kaneno et al., 2004). This shows that application
of microreactors with immobilized lipases

Table 2
Model enzymatic systems involving microreactors
Enzyme Medium Application Refs.
Acetylcholinesterase, urease Poly(ethylene glycol) hydrogel Biotransformation in
hydrogel arrays
Yadavalli et al., 2004
Alanine aminotransferase, Sieved porous glass beads Determination of l-alanine,
Janasek and Spohn, 1999
glutamate oxidase
a-ketoglutarate and l-glutamate
Alkaline phosphatase Glass slide Development of microscale
steady-state kinetic analysis
Gleason and Carbeck, 2004
Alkaline phosphatase
and bienzymatic system
(glucose oxidase and
horseradish peroxidase)
Alkaline phosphatase
and bienzymatic system
(glucose oxidase
and peroxidase)
Alkaline phosphatase,
glucose oxidase,
horseradish peroxidase
Poly(dimethylsiloxane)/glass Method for photopatterning
well-defined patches of enzymes
inside a microfluidic device
Nitrocellulose membrane on glass Method for immobilizing
enzymes without chemical
modification of a microchannel
Phospholipid bilayers inside
poly(dimethylsiloxane)
microchannels
Rapid determination of
enzyme kinetics at many
different substrate concentrations
by carrying out laminar flowcontrolled
dilution on-chip
pH-sensitive fluorophore
used to monitor changes of pH
Holden et al., 2004
Park et al., 2003
Mao et al., 2002
Alkaline phosphatase, urease Hydrogel copolymerized
with enzymes
Koh and Pishko, 2005
Ascorbate oxidase Silicon Glutamate monitoring Collins et al., 2001
Aatalase, penicillinase Silicon wafer Sensing of microlitre samples Xie et al., 1992
Cucumisin, l-lactic
Fused-silica capillary Development of immobilization
Miyazaki et al., 2004
dehydrogenase
techniques
Different enzymes Polydimethylsiloxane cast Direct incorporation of the
Jones et al., 2002
on silicon/SU-8 moulds
enzyme onto the wall material
Glucose dehydrogenase Streptavidin-coated
Study of PQQ-dependent
Zhao and Wittstock, 2004
paramagnetic beads
quinoprotein (use of scanning
electrochemical microscopy)
Glucose oxidase Aminopropyl controlledpore
glass particles
Determination of glucose Xu and Fang, 2004
Glucose oxidase Controlled-pore glass Determination of glucose Strike et al., 2000
Glucose oxidase Controlled-pore glass beads Determination of codeine
and glucose
L’Hostis et al., 2000
Glucose oxidase Enzyme-immobilized
magnetic microparticles
Glucose measurement by FIA Nomura et al., 2004
Glucose oxidase Fused silica l-glutamate monitoring Niwa et al., 1999
Glucose oxidase Porous silicon Glucose monitoring Drott et al., 1997
Glucose oxidase Porous silicon Influence of the matrix
depth investigated
Drott et al., 1999
Glucose oxidase Silica Determination of glucose Kulys, 1999
Glucose oxidase Silicon chip Glucose measurement in flow Murakami et al., 1993
Glucose oxidase Silicon wafer Continuous glucose monitoring
in a microdialysis-based system
Laurell et al., 1995
Glucose oxidase,
Porous silicon Glucose and glutamate monitoring, Bengtsson et al., 2000
ascorbate oxidase, trypsin
myoglobin cleavage
Glucose oxidase,
Hydrogel copolymerized
Determination of glucose Zhan et al., 2002
horseradish peroxidase with enzymes
Glucose oxidase,
Polycrystalline gold and glass Formation of micropatterns
Wilhelm and Wittstock, 2002
horseradish peroxidase
of enzymes
Glucose oxidase, invertase Glass-supported aminopropyl Determination of glucose
and sucrose
Folly et al., 1997
Horseradish peroxidase Sapphire wafer Use of homovanillic acid
fluorescence assay
Heule et al., 2003
Horseradish peroxidase,
uricase
Sol–gel Determination of uric acid Lv et al., 2003
(continued on next page)

Table 2 (continued)
Enzyme Medium Application Refs.
Horseradish peroxidase, Microbeads Measurement of enzyme kinetics Seong et al., 2003
h-galactosidase
(fluorescence imaging)
Lipase Glass beads, alginate gel
Hydrolysis of triacetin,
Pijanowska et al., 2001
beads, nitrocellulose sheets tributyrin and triolein
Lipase SiO2-coated microcapillary Hydrolysis of umbelliferone acetate Nakamura et al., 2004
Lipases, proteases,
glucose oxidase,
horseradish peroxidase
Sol–gel arrays Screening of proteins (enzymes) Park and Clark, 2002
Peroxidase, glucose oxidase Silicon wafer Continuous glucose measurements Laurell and Rosengren, 1994
PikC hydroxylase Ni-NTA agarose beads Rapid hydroxylation of macrolides Srinivasan et al., 2004
Protease Silica monolith Transesterification (glycidol, n-butyrate) Kawakami et al., 2005
Trehalase Aminopropyl glass particles Quantification of trehalose Bachinski et al., 1997
Urease Polydimethylsiloxane High urea conversion in continuous flow Jones et al., 2004
B-Fructosidase Porous silicon Determination of sucrose Lendl et al., 1997
offers a great advantage by shortening the analysis time.
In batch reactions, completion of enzyme-catalysed
transesterification may take days for some supported
lipases (Kamal et al., 2002).
On account of reproducible distribution of the products
formed along the axes of the microreactor, the
enzymatic process can be visualized by fluorescence
microscopy in order to acquire data on the concentration
patterns inside the device. Such an approach was presented
by Seong et al. (2003), and fluorescence images
of the reaction zone together with scaled numerical
results from the cross-sections of input and output
streams are shown in Fig. 4. The method provided
high sensitivity for product detection and short response
time, and kinetic graphs of the reaction catalyzed by the
enzyme (horseradish peroxidase) were obtained. The
Lilly–Hornby model was used to characterize the kinet-
ics of biocatalysis in the packed microcolumn, and
results were compared with those for kinetics of the
enzyme in homogeneous solution. The Michaelis constants
were found to be similar to those obtained from
the Lineweaver–Burke model for the homogeneous catalysis.
In comparison with standard assays, the amount
of enzyme used was very small: Seong et al. (2003)
estimated that 200 pmol (3 10 9 molecules of enzyme)
were required for the analysis. The current trend in
biochemical analysis is to decrease the amount of biocatalyst
used. Recently, Moore et al. (2004) presented an
assay for 500 lipase molecules, capable of application to
single cells. Rondelez et al. (2005) described an assay
for monitoring reaction catalyzed by a single molecule
of h-galactosidase and horseradish peroxidase.
The kinetics model described by Lilly et al. (1966) is
appropriate for systems with continuous flow of the
Fig. 3. Schematic for continuous-flow reaction and monitoring of hydrolysis of esters using microreactor packed with lipase immobilized onto either
nitrocellulose sheets or glass beads coated with keratin. Reprinted from Pijanowska et al. (2001) with permission from Elsevier.

Fig. 4. (A) Horse radish peroxidase-catalyzed reaction between non-fluorescent amplex red and H2O2 to yield fluorescent resorufin. (B)
Fluorescence micrograph of the microreactor during continuous-flow operation. The substrate solution contained 5 AM H2O2 and 10 AM amplex
red in 50 mM Tris-HCl buffer (pH 7.4) and was introduced into the microreactor from left to right. Flow rate, 0.5 Al min -1 . (C) Normalized
fluorescence intensity line scans obtained at locations indicated by the dashed lines in (B). The excitation and maximum emission wavelengths were
563 and 587 nm, respectively. Reprinted with permission from Seong et al. (2003). Copyright (2003) American Chemical Society.
substrate and under steady-state conditions, and can be
summarized by the following equation:
Ps0 ¼ K V m lnð1PÞþC=Q ð1Þ
where P is the fraction of substrate reacted in the
column, s0 the substrate concentration at the beginning,
KVm the apparent Michaelis constant, C the reaction
capacity of the reactor, and Q the flow rate of the
substrate. This formula allows determination of the
apparent Michaelis constant of the catalytical process
when all other parameters are known. If any masstransfer
effects contribute to dynamics, an extrapolation
to zero flow rate is required to obtain the value of the
Michaelis constant for comparison with that of free
enzyme (Seong et al., 2003).
Mass transfer is always an important issue when
considering enzymes entrapped in supports, and the
ideal situation is when diffusion of substrate and product
into and out of the bulk solution is not the rate
limiting process. Koh and Pishko (2005) determined
Michaelis constants of enzymes entrapped in hydrogel
micropatches in microfluidic channels using Lineweaver–Burke
graphs. Values were found to be lower, by
approximately an order of magnitude, than those
obtained from experiments using the homogeneous
enzymes. The influence of entrapment in the hydrogel
nanostructure on the kinetic properties of the enzymes
was discussed.
2.2.2. Imaging of biotransformations in microreactors
Apart from the study described in Section 2.2.1.
(Seong et al., 2003), up to now there have been few
other attempts to image the enzymatic reaction zone. In
the continuous flow mode, imaging allows comparison
of the signals at inlet and outlet of the microreactor, and
hence data on the rate of substrate conversion in a
single pass of the reactor. Koh and Pishko (2005)
used seminaphthofluorescein (SNAFL-1) as a pH indicator
and enzymes copolymerized with poly(ethylene
glycol) to monitor biocatalytic processes resulting in a
change of acidity. Variations in the fluorescence intensity
were monitored by fluorescence microscopy during

the hydrolysis of p-nitrophenylphosphate and urea catalyzed
by alkaline phosphatase and urease, respectively.
Yadavalli et al. (2004) presented a microarray-based
system with immobilized enzymes which enabled
screening of low concentrations of enzyme substrates.
The hydrogel arrays were prepared photolithographically
on silicon surfaces. In earlier work, Zhan et al.
(2002) developed a similar method for the monitoring
of the reactions catalyzed by glucose oxidase and
horseradish peroxidase entrapped in a hydrogel matrix
(Fig. 5). The oxidation of glucose was followed by
decomposition of Amplex red dye (a substrate of horseradish
peroxidase) which resulted in formation of fluorescent
resorufin. It was concluded that the pores in the
hydrogels were sufficiently small to retain the enzymes
and the reporter dyes, and it was suggested that the
method could be applied to immobilization and monitoring
of reactions of other proteins.
Recently Rondelez et al. (2005) managed to follow
the reaction catalyzed by single molecules of h-galactosidase
and horseradish peroxidase entrapped between
glass/PDMS slides, in a non-microfluidic system. This
was facilitated by a fluorescence assay and watching
the product detection within a few minutes.
Any kind of biocatalysis imaging in enzymatic
microreactors is dependent on the existence of appropriate
assays, usually involving fluorescence spectrometry.
UV-Vis imaging is possible in principle, but would
require the use of channels and supports made of a
material such as silica which is transparent over the
wavelength range used.
Fig. 5. Micrographs of hydrogel micropatches within a microfluidic
channel: (A) optical micrograph; (B) fluorescence micrograph of the
same micropatches shown in (A). Fluorescence arises from the dye
SNAFL-1 entrapped within the hydrogel. Reprinted with permission
from Zhan et al. (2002). Copyright (2002) American Chemical
Society.
3. Conclusions and future trends
So far, very few enzymes have been applied within
microreactors, although it seems the new devices will be
developed not only as model systems but they will also
be directed to specific problems, as already happens in
the case of tryptic digestion and PCR microreactors.
There are few published patents describing construction
of enzymatic microreactors (Fujii and Hosokawa, 1998;
Combette and Constantin, 2003; Miyazaki and Maeda,
2004a,b), which indicates that developments of applications
in this field are still in the initial stage.
One applications-oriented example of use of enzymatic
microreactors is the hydrolysis of used grease and
its conversion to diesel fuel (Hsu et al., 2002). This also
points to the bgreenQ aspects of microreactors, due to
their low maintenance requirements, as well as applications
in environmental protection. There is a huge commitment
by the pharmaceutical industry to the search for
new potent inhibitors of lipases, that can be employed in
the treatment of obesity (Müller and Petry, 2004), and
fast analytical procedures for these biocatalysts are required.
A variety of immobilized lipases available from
a range of suppliers (e.g. Sigma Aldrich, Amano, Bio-
Chemika, Novozymes) may be used in microsystems
produced for fast screening of inhibitors of these
enzymes. Other immobilized enzymes are already
used in industrial syntheses (Buchholz et al., 2005).
Various aspects of enzyme immobilization including
stability issues have been discussed by Cao (2005).
Enzymatic microreactors have the potential for introduction
into industrial-scale synthesis. They can be
easily incorporated in systems operating in the external
numbering-up mode, where the reaction subunits are
cased separately and put together externally. This
mode of scaling up reactions provides good adjustability
and control over the process, due to repetition of the
fluidic path while the transport properties and hydrodynamics
are preserved (Hessel et al., 2004). Any microreactor
units containing enzyme found to be lacking
sufficient activity can be easily replaced with new
ones, with minimal effects on the performance of the
whole system.
The sine qua non-condition for any large scale use of
enzymatic microreactors is of ease of use and robustness,
together with commercialization of microsystem
components. Robustness in part governed by the enzyme
stability, and lipases have the advantages of
stability at ambient temperatures whether immobilized
and stored dry or in an organic solvent.
The other important issue is setting up the interface
systems to operate the microreactors. Such systems

involve sampling, injection, flow control (e.g. pumping)
and monitoring of the product. Many of these steps
can be realized by incorporating enzymes in the structure
of lab-on-a-chip which then can be integrated
within portable analytical devices. Nevertheless, appropriate
interfacing between macro and micro instruments
is indispensable (Fredrickson and Fan, 2004). Microreactors
have already been used within analytical
instruments, e.g. capillary electrophoresis, high performance
liquid chromatography and mass spectrometry.
Fabrication of such systems on the industrial scale will
facilitate parallel analyses and obviate the need for
construction of home-made set-ups with syringe
pumps and capillary detectors.
High resolution screening (HRS) is a generic term
for methods based on high performance separation of a
mixture of compounds followed by on-line affinity
recognition, usually achieved with an enzymatic assay
and MS detection. HRS is an advantageous technique
in drug discovery (Irth et al., 2004) since it enables
evaluation of their affinity for target enzymes. Whilst
the enzyme is usually injected into the system following
separation of the mixture components using HPLC, an
alternative is to connect a microreactor with an immobilized
enzyme to the output of an HPLC column.
These approaches facilitate investigation of the action
of enzymes towards new drug candidates, and other
targets in analysis and synthesis (Girelli and Mattei,
2005). Ma et al. (2000) demonstrated a system where
the enzyme (lactate dehydrogenase) was injected into
96 wells, incubated (30–1477 min), and the contents of
the wells sampled and separated by capillary electrophoresis
in 96 multiplexed capillaries. This parallel
assay approach allowed rapid evaluation of the effects
of change of pH and enzyme concentration, and optimization
of reaction conditions.
There is an undoubted need for development of
microreactors and multiplexed methodologies with all
the enzymes which are frequently used, especially in
biochemical enzymatic methods, so that optimization
can be performed quickly. A desirable goal is for highthroughput
screening of enzymes, their substrates and
inhibitors. Use of microreactors with parallel microfluidic
streams could facilitate the selection from an array
of enzymes of a specific enzyme for optimum transformation
of the chosen substrate. Prospective fields of
application of microreactors are quite wide and include
biotechnology, as well as combinatorial chemistry and
enzyme-targeted drug search.
This review of the recent development of enzymatic
reaction technology has shown that such miniaturized
systems find applications in many fields, especially in
the analysis of proteins and nucleic acids. The development
of this field is in part limited by the availability
of enzymes immobilized on solid supports, as well as
by the range of assays that permit monitoring progress
of the reactions on a microscale. Use of microreactors
also facilitates kinetic studies of immobilized enzymes,
using extremely small quantities of biocatalyst material.
Since supported enzymes can be used in continuous
biocatalytic processes, they have the potential to replace
homogeneous catalysis protocols. Whilst most of
the examples given in this review are of research
applications, adoption of the techniques in standard
analytical and micropreparative procedures would undoubtedly
be aided by the commercialization of enzymatic
microreactors.
Acknowledgements
The authors would like to acknowledge the financial
support of the European Community received as a part of
the project bCHEMCELL: Chemical Biology in Reactors
and CellsQ (Contract No. MEST-CT-2004-504345).
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