Enzymatic microreactors in chemical analysis and kinetic studies

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