Enzymatic microreactors in chemical analysis and kinetic studies

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