Enzymatic microreactors in chemical analysis and kinetic studies

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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 substrate, 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.
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