Biotechnology Advances 24 (2006) 42 – 57 www.elsevier.com/locate/biotechadv Research review paper Enzymatic microreactors in chemical analysis and kinetic studies Pawel L. Urban a, David M. Goodall a,*, Neil C. Bruce b a b Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK 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 Abstract 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. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of enzymatic microreactors . . . . . . . . . . . . 2.1. Analysis of chemical species. . . . . . . . . . . . . . . 2.1.1. Homogeneous and heterogeneous biocatalysis . 2.1.2. Analysis of proteins . . . . . . . . . . . . . . . 2.1.3. Analysis of nucleic acids . . . . . . . . . . . . 2.1.4. Model enzymatic systems . . . . . . . . . . . . 2.2. Other applications . . . . . . . . . . . . . . . . . . . . 2.2.1. Kinetic studies. . . . . . . . . . . . . . . . . . 2.2.2. Imaging of biotransformations in microreactors . 3. Conclusions and future trends. . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * Corresponding author. Tel.: +44 1904 435 328; fax: +44 1904 432 516. E-mail address: dmg1@york.ac.uk (D.M. Goodall). 0734-9750/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2005.06.001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 44 44 44 44 46 48 48 48 51 52 53 53 P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 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- 43 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 44 P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 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. 45 P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 Table 1 Application of enzymatic microreactors in the analysis of proteins Enzyme Medium Application Reference Chymotrypsin Chymotrypsin, trypsin, papain Silicon Magnetic microparticles Ekström et al., 2000 Korecka et al., 2004 Pepsin Gel on a photopolymerized porous silica monolith Protease Protease Trypsin Fused-silica capillary (metal–ion chelated adsorption ) Porous wall of a capillary Controlled pore glass Trypsin Trypsin Controlled pore glass Fused-silica capillary Trypsin Trypsin Fused-silica capillary Fused-silica capillary Trypsin Trypsin Peptide mapping Jiang et al., 2000b On-line frontal analysis of enzymatic products Digestion of insulin Protein patterning Jiang et al., 2000a Trypsin Gel beads Glycidyl methacrylatemodified cellulose membrane Glycidyl methacrylatemodified cellulose membrane Glycidyl methacrylatemodified cellulose Injected Micropatterned sol–gel structures in polydimethylsiloxane microchannels Monolith Protein identification Specific fragmentation of high molecular-mass and heterogeneous glycoproteins Protein digestion, peptide separation, and protein identification Peptide mapping analysis of proteins Protein mapping On-line protein digestion, preconcentration, separation and detection (in UV) Peptide mapping Analysis of proteins and peptides Digestion of proteins On-column digestions of small amounts of proteins Digestion of proteins Peptide mapping Peterson et al., 2003 Trypsin Trypsin Monolith Monolithic capillary column Trypsin Trypsin Trypsin Porous polymer monolith Porous polymer monolith Porous polymer monolith in fused-silica capillary Porous silicon Enzyme digestion for peptide mapping Analysis of proteins Digestion of picomoles of proteins Protein mapping Peptide mass mapping Peptide mass mapping Bengtsson et al., 2002 Jiang and Lee, 2001 Trypsin Trypsin Trypsin Trypsin Trypsin, glucose oxidase Trypsin Trypsin PVDF membrane disk Trypsin Trypsin Trypsin and pepsin (proteases) Trypsin RP beads Silica gel microchannels Fused-silica capillary High-speed on-line protein digestion Protein mapping (lactate dehydrogenase) Extraction of proteins from 2D gels and digestion Protein mapping Proteomic research Peptide mapping poly(vinylidene fluoride) in Poly(dimethylsiloxane) channel Rapid protein digestion, peptide separation, and protein identification Porozyme One example of such a system involves a homemade microreactor with trypsin immobilized on controlled pore glass (CPG) beads (Bonneil et al., 2000), Kato et al., 2004 Guo et al., 2003 Guo et al., 2002 Bonneil and Waldron, 2000 Bonneil et al., 2000 Licklider et al., 1995 Licklider and Kuhr, 1998 Amankwa and Kuhr, 1992 Jin et al., 2003 Jiang et al., 2000c Gottschlich et al., 2000 Kim et al., 2001 Xie et al., 1999 Ye et al., 2004 Peterson et al., 2002a Palm and Novotny, 2004 Peterson et al., 2002b Samskog et al., 2003 Cooper and Lee, 2004 Ekström et al., 2002 Qu et al., 2004 Licklider and Kuhr, 1994 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- 46 P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 Fig. 1. Set-up for digestion in the immobilized-trypsin microreactor. Reprinted from Bonneil et al. (2000) with permission from Elsevier. ing the trypsin-CPG beads. A low pressure of argon gas was applied to drive the buffer solution containing a protein sample through the capillary. The flow rate in the system was calibrated by collecting and weighing the amount of solution emerging from the microreactor in a measured time. The system could be constructed in 30 min. A peptide map of h-casein was obtained within the total time of 3 h (digestion and CE separation). Microreactors can be easily attached to the nebuliser for an electrospray ionisation (ESI) interface to mass spectrometry (MS), providing an on-line system. Alternatively, the product fractions may be sampled prior to off-line analysis by means of matrix assisted laser desorption ionisation — time of flight (MALDITOF)-MS. Although trypsin is the most frequently used catalyst for protein hydrolysis, other proteases (e.g. chymotrypsin, pepsin and papain) may also be utilised. The enzymes are immobilized on a variety of media, either particles filling the microreactor cavity, or bound to the inner walls of the capillary (Nashabeh and Rassi, 1992; Verpoorte, 2003a). In the latter case, there are no backpressure constraints during sample injection. Enzyme immobilization on the walls of fused-silica capillaries is also convenient because such capillaries are widely available and inexpensive, and because electrophoretic separation can be carried out in the same capillary as the enzymatic reaction. This approach can in principle reduce systematic errors associated with moving the sample from the microreactor into the separation zone. 2.1.3. Analysis of nucleic acids A notable achievement of microtechnology has been setting up systems with microreactors for polymerase chain reaction (PCR), enabling automation of DNA amplification. This has allowed the construction of high throughput systems for fast analysis of genetic material. Typically, the substrates for PCR reaction and polymerase enzyme are injected into the reaction zone (Zhang and Yeung, 1998; Khandurina et al., 2000; Lee et al., 2000; Nagai et al., 2001; Schneegass et al., 2001; Ke et al., 2004), and a thermal cycling P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 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 47 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. 48 P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 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, K m and v max, to be 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 49 P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 Table 2 Model enzymatic systems involving microreactors Enzyme Medium Application Refs. Acetylcholinesterase, urease Poly(ethylene glycol) hydrogel Yadavalli et al., 2004 Alanine aminotransferase, glutamate oxidase Alkaline phosphatase Sieved porous glass beads Biotransformation in hydrogel arrays Determination of l-alanine, a-ketoglutarate and l-glutamate Development of microscale steady-state kinetic analysis Method for photopatterning well-defined patches of enzymes inside a microfluidic device Glass slide 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 Alkaline phosphatase, urease Hydrogel copolymerized with enzymes Silicon Silicon wafer Fused-silica capillary Ascorbate oxidase Aatalase, penicillinase Cucumisin, l-lactic dehydrogenase Different enzymes Glucose dehydrogenase Glucose oxidase Glucose oxidase Glucose oxidase Glucose oxidase Janasek and Spohn, 1999 Gleason and Carbeck, 2004 Holden et al., 2004 Nitrocellulose membrane on glass Method for immobilizing enzymes without chemical modification of a microchannel Park et al., 2003 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 Glutamate monitoring Sensing of microlitre samples Development of immobilization techniques Direct incorporation of the enzyme onto the wall material Study of PQQ-dependent quinoprotein (use of scanning electrochemical microscopy) Determination of glucose Mao et al., 2002 Determination of glucose Determination of codeine and glucose Glucose measurement by FIA Strike et al., 2000 L’Hostis et al., 2000 l-glutamate monitoring Glucose monitoring Influence of the matrix depth investigated Determination of glucose Glucose measurement in flow Continuous glucose monitoring in a microdialysis-based system Glucose and glutamate monitoring, myoglobin cleavage Determination of glucose Niwa et al., 1999 Drott et al., 1997 Drott et al., 1999 Polydimethylsiloxane cast on silicon/SU-8 moulds Streptavidin-coated paramagnetic beads Aminopropyl controlledpore glass particles Controlled-pore glass Controlled-pore glass beads Glucose oxidase Glucose oxidase Glucose oxidase Enzyme-immobilized magnetic microparticles Fused silica Porous silicon Porous silicon Glucose oxidase Glucose oxidase Glucose oxidase Silica Silicon chip Silicon wafer Glucose oxidase, ascorbate oxidase, trypsin Glucose oxidase, horseradish peroxidase Glucose oxidase, horseradish peroxidase Glucose oxidase, invertase Porous silicon Glass-supported aminopropyl Horseradish peroxidase Sapphire wafer Horseradish peroxidase, uricase Sol–gel Hydrogel copolymerized with enzymes Polycrystalline gold and glass Formation of micropatterns of enzymes Determination of glucose and sucrose Use of homovanillic acid fluorescence assay Determination of uric acid Koh and Pishko, 2005 Collins et al., 2001 Xie et al., 1992 Miyazaki et al., 2004 Jones et al., 2002 Zhao and Wittstock, 2004 Xu and Fang, 2004 Nomura et al., 2004 Kulys, 1999 Murakami et al., 1993 Laurell et al., 1995 Bengtsson et al., 2000 Zhan et al., 2002 Wilhelm and Wittstock, 2002 Folly et al., 1997 Heule et al., 2003 Lv et al., 2003 (continued on next page) 50 P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 Table 2 (continued) Enzyme Medium Application Refs. Horseradish peroxidase, h-galactosidase Lipase Microbeads Seong et al., 2003 Glass beads, alginate gel beads, nitrocellulose sheets SiO2-coated microcapillary Sol–gel arrays Measurement of enzyme kinetics (fluorescence imaging) Hydrolysis of triacetin, tributyrin and triolein Hydrolysis of umbelliferone acetate Screening of proteins (enzymes) Silicon wafer Ni-NTA agarose beads Silica monolith Aminopropyl glass particles Polydimethylsiloxane Porous silicon Continuous glucose measurements Rapid hydroxylation of macrolides Transesterification (glycidol, n-butyrate) Quantification of trehalose High urea conversion in continuous flow Determination of sucrose Laurell and Rosengren, 1994 Srinivasan et al., 2004 Kawakami et al., 2005 Bachinski et al., 1997 Jones et al., 2004 Lendl et al., 1997 Lipase Lipases, proteases, glucose oxidase, horseradish peroxidase Peroxidase, glucose oxidase PikC hydroxylase Protease Trehalase Urease B-Fructosidase 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- Pijanowska et al., 2001 Nakamura et al., 2004 Park and Clark, 2002 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
109 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. P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 51 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 KmV lnð1  PÞ þ C=Q ð1Þ where P is the fraction of substrate reacted in the column, s 0 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 52 P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 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, BioChemika, 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 P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 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 53 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). References Amankwa LN, Kuhr WG. Trypsin-modified fused-silica capillary microreactor for peptide-mapping by capillary zone electrophoresis. Anal Chem 1992;64:1610 – 3. Avila LZ, Whitesides GM. Catalytic activity of native enzymes during capillary electrophoresis — an enzymatic microreactor. J Org Chem 1993;58:5508 – 12. Bachinski N, Martins AS, Paschoalin VMF, Panek AD, Paiva CLA. Trehalase immobilization on aminopropyl glass for analytical use. Biotechnol Bioeng 1997;54:33 – 9. Bao J, Regnier FE. Ultramicro enzyme assays in a capillary electrophoretic system. J Chromatogr 1992;608:217 – 24. Bengtsson M, Ekström S, Drott J, Collins A, Csoregi E, Marko-Varga G, et al. Applications of microstructured porous silicon as a biocatalytic surface. Phys Status Solidi, A Appl Res 2000;182:495 – 504. Bengtsson M, Ekström S, Marko-Varga G, Laurell T. Improved performance in silicon enzyme microreactors obtained by homogeneous porous silicon carrier matrix. Talanta 2002; 56:341 – 53. Bilitewski U, Genrich M, Kadow S, Mersal G. Biochemical analysis with microfluidic systems. Anal Bioanal Chem 2003; 377:556 – 69. Bonneil E, Waldron KC. On-line system for peptide mapping by capillary electrophoresis at sub-micromolar concentrations. Talanta 2000;53:687 – 99. Bonneil E, Mercier M, Waldron KC. Reproducibility of a solid-phase trypsin microreactor for peptide mapping by capillary electrophoresis. Anal Chim Acta 2000;404:29 – 45. Buchholz K, Kasche V, Bornscheuer UT. Biocatalysts and enzyme technology. Weinheim7 Wiley-VCH; 2005. Burns MA, Johnson BN, Brahmasandra SN, Handique K, Webster JR, Krishnan M, et al. An integrated nanoliter DNA analysis device. Science 1998;282:484 – 7. 54 P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 Cao L. Immobilized enzymes: science or art? Curr Opin Chem Biol 2005;9:217 – 26. Carvalho CML, Cabral JMS. Reverse micelles as reaction media for lipases. Biochimie 2000;82:1063 – 85. Chiesl TN, Shi W, Barron AE. Poly(acrylamide-co-alkylacrylamides) for electrophoretic DNA purification in microchannels. Anal Chem 2005;77:772 – 9. Chopineau J, Robert S, Fénart L, Cecchelli R, Lagoutte B, Paitier S, et al. Monoacylation of ribonuclease A enables its transport across an in vitro model of the blood-brain barrier. J Control Release 1998;56:231 – 7. Collins A, Mikeladze E, Bengtsson M, Kokaia M, Laurell T, Csöregi E. Interference elimination in glutamate monitoring with chip integrated enzyme microreactors. Electroanalysis 2001;13:425 – 31. Combette, Ph., Constantin, O., 2003. Microreactor, method for preparing same, and method for producing a biochemical or biological reaction. Patent No. WO03097229. Cooper JW, Lee CS. Integrated and ultrasensitive gel protein identification. Anal Chem 2004;76:2196 – 202. de Mello AJ, Beard N. Dealing with drealT samples: sample pretreatment in microfluidic systems. Lab Chip 2003;3:11N – 9N. Drott J, Lindstrom K, Rosengren L, Laurell T. Porous silicon as the carrier matrix in microstructured enzyme reactors yielding high enzyme activities. J Micromech Microeng 1997;7:14 – 23. Drott J, Rosengren E, Lindstrom K, Laurell T. Porous silicon carrier matrices in micro enzyme reactors — influence of matrix depth. Microchim Acta 1999;131:115 – 20. Ehrfeld W, Hessel V, Löwe H. Microreactors. Weinheim7 WileyVCH; 2000. Ekström S, Önnerfjord P, Nilsson J, Bengtsson M, Laurell T, Marko-Varga G. Integrated microanalytical technology enabling rapid and automated protein identification. Anal Chem 2000;72:286 – 93. Ekström S, Malmström J, Wallman L, Löfgren M, Nilsson J, Laurell T, et al. On-chip microextraction for proteomic sample preparation of in-gel digests. Proteomics 2002;2:413 – 21. Fang Q. Sample introduction for microfluidic systems. Anal Bioanal Chem 2004;378:49 – 51. Folly R, Salgado A, Valdman B, Valero F. The development of enzymatic sensors for the continuous monitoring of glucose and sucrose. Braz J Chem 1997;14. Fredrickson CK, Fan ZH. Macro-to-micro interfaces for microfluidic devices. Lab Chip 2004;4:526 – 33. Fujii, T., Hosokawa, K., 1998. Microreactor for biochemical reaction. Patent No. JP10337173. Garti N. Microemulsions as microreactors for food applications. Curr Opin Colloid Interface Sci 2003;8:197 – 211. Garti N, Lichtenberg D, Silberstein T. The hydrolysis of phosphatidylcholine by phospholipase A2 in microemulsion as microreactor. Colloids Surf, A Physicochem Eng Asp 1997;128:17 – 25. Girelli AM, Mattei E. Application of immobilized enzyme reactor in on-line high performance liquid chromatography: a review. J Chromatogr B 2005;819:3 – 16. Gleason NJ, Carbeck JD. Measurement of enzyme kinetics using microscale steady-state kinetic analysis. Langmuir 2004;20: 6374 – 81. Gottschlich N, Culbertson CT, McKnight TE, Jacobson SC, Ramsey JM. Integrated microchip-device for the digestion, separation and postcolumn labeling of proteins and peptides. J Chromatogr B 2000;745:243 – 9. Guijt-van Duijn RA, Moerman R, Kroon A, van Dedem GWK, van den Doel R, van Vliet L, et al. Miniaturized analytical assays in biotechnology. Biotechnol Adv 2003;21:431 – 44. Guo Z, Zhang QC, Lei ZD, Kong L, Mao XQ, Zou HF. Studies on rapid micro-scale peptide mapping analysis using a capillary micro-reactor. Chem J Chin Univ — Chin 2002;23: 1277 – 80. Guo Z, Xu SY, Lei ZD, Zou HF, Guo BC. Immobilized metalion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ionizationtime of flight-mass spectrometry. Electrophoresis 2003;24: 3633 – 9. Hashimoto M, He Y, Yeung ES. On-line integration of PCR and cycle sequencing in capillaries: from human genomic DNA directly to called bases. Nucleic Acids Res 2003;31:41. Hashimoto M, Chen PC, Mitchell MW, Nikitopoulos DE, Soper SA, Murphy MC. Rapid PCR in a continuous flow device. Lab Chip 2004;4:638 – 45. Haswell SJ, Skelton V. Chemical and biochemical microreactors. Trends Anal Chem 2000;19:389 – 95. Haswell SJ, Watts P. Green chemistry: synthesis in micro reactors. Green Chem 2003;5:240 – 9. Hessel V, Hardt S, Löwe H. Chemical micro process engineering. Weinheim7 Wiley-VCH; 2004. Hessel V, Hardt S, Löwe H, Müller A, Kolb G. Chemical Micro Process Engineering. Weinheim7 Wiley-VCH; 2005a. Hessel V, Löwe H, Müller A, Kolb G. Chemical micro process engineering. Processing and plants. Weinheim7 Wiley-VCH; 2005b. Heule M, Rezwan K, Cavalli L, Gauckler LJ. A miniaturized enzyme reactor based on hierarchically shaped porous ceramic microstruts. Adv Mater 2003;15:1191 – 4. Holden MA, Jung SY, Cremer PS. Patterning enzymes inside microfluidic channels via photo attachment chemistry. Anal Chem 2004;76:1838 – 43. Hsu A-F, Jones K, Foglia TA, Marmer WN. Immobilized lipasecatalysed production of alkyl esters of restaurant grease as biodiesel. Biotechnol Appl Biochem 2002;36:181 – 6. Irth H, Long S, Schenk T. High-resolution screening in an expanded chemical space. Curr Drug Discov 2004:19 – 23. Janasek D, Spohn U. Chemiluminometric flow injection analysis procedures for the enzymatic determination of l-alanine, alphaketoglutarate and l-glutamate. Biosens Bioelectron 1999;14: 123 – 9. Jiang Y, Lee CS. On-line coupling of micro-enzyme reactor with micro-membrane chromatography for protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry. J Chromatogr A 2001;924:315 – 22. Jiang H, Zou H, Wang H, Ni J, Zhang Q, Zhang Y. On-line characterization of the activity and reaction kinetics of immobilized enzyme by high-performance frontal analysis. J Chromatogr A 2000a;903:77 – 84. Jiang HH, Zou HF, Wang HL, Ni JY, Zhang Q. Covalent immobilization of trypsin on glycidyl methacrylate-modified cellulose membrane as enzyme reactor. Chem J Chin Univ — Chin 2000b;21:702 – 6. Jiang HH, Zou HF, Wang HL, Zhang Q, Ni JY, Zhang QC, et al. Combination of MALDI-TOF mass spectrometry with immobilized enzyme microreactor for peptide mapping. Sci China, Ser B Chem Life Sci Earth Sci 2000c;43:625 – 33. Jianrong Ch, Yuqing M, Nongyue H, Xiaohua W, Sijiao L. Nanotechnology and biosensors. Biotechnol Adv 2004;22:505 – 18. P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 Jin LJ, Ferrance J, Sanders JC, Landers JP. A microchip-based proteolytic digestion system driven by electroosmotic pumping. Lab Chip 2003;3:11 – 8. Jones F, Lu ZH, Elmore BB. Development of novel microscale system as immobilized enzyme bioreactor. Appl Biochem Biotechnol 2002;98:627 – 40. Jones F, Forrest S, Palmer J, Lu ZH, Elmore J, Elmore BB. Immobilized enzyme studies in a microscale bioreactor. Appl Biochem Biotechnol 2004;113:261 – 72. Kamal A, Sandbhor M, Ramana KV. One-pot lipase-catalyzed synthesis of enantiopure secondary alcohols from carbonyl compounds: a new protocol for lipase-mediated resolution. Tetrahedron: Asymmetry 2002;13:815 – 20. Kan CW, Fredlake CP, Doherty EAS, Barron AE. DNA sequencing and genotyping in miniaturized electrophoresis systems. Electrophoresis 2004;25:3564 – 88. Kaneno J, Kohama R, Miyazaki M, Uehara M, Kanno K, Fujii M, et al. Development of surface modification method and its application for preparation of enzyme-immobilized microreactor. Kagaku Kogaku Ronbunshu 2004;30:154 – 8. Kanie Y, Kanie O. Electrophoretically mediated microanalysis: enzyme analyses at a nanoliter scale. Beckman Coulter PACE Setter 2003;7:1 – 4. Kanno K, Maeda H, Izumo Sh, Ikuno M, Takeshita K, Teshiro A, et al. Rapid enzymatic transglycosylation and oligosaccharide synthesis in a microchip reactor. Lab Chip 2002;2:15 – 8. Kato M, Sakai-Kato K, Jin HM, Kubota K, Miyano H, Toyo’oka T, et al. Integration of on-line protein digestion, peptide separation, and protein identification using pepsin-coated photopolymerized sol– gel columns and capillary electrophoresis/mass spectrometry. Anal Chem 2004;76:1896 – 902. Katsura Sh, Harada N, Maeda Y, Komatsu J, Matsuura Sh, Takashima K, et al. Activation of restriction enzyme by electrochemically released magnesium ion. J Biosci Bioeng 2004;98:293 – 7. Kawakami K, Sera Y, Sakai Sh, Ono T, Ijima H. Development and characterization of a silica monolith immobilized enzyme microbioreactor. Ind Eng Chem Res 2005;44:236 – 40. Ke C, Berney H, Mathewson A, Sheehan MM. Rapid amplification for the detection of Mycobacterium tuberculosis using a noncontact heating method in a silicon microreactor based thermal cycler. Sens Actuators, B, Chem 2004;102:308 – 14. Khandurina J, McKnight TE, Jacobson SC, Waters LC, Foote RS, Ramsey JM. Integrated system for rapid PCR-based DNA analysis in microfluidic devices. Anal Chem 2000;72:2995 – 3000. Kim YD, Park CB, Clark DS. Stable sol–gel microstructured and microfluidic networks for protein patterning. Biotechnol Bioeng 2001;73:331 – 7. Koh W-G, Pishko M. Immobilization of multi-enzyme microreactors inside microfluidic devices. Sens Actuators, B, Chem 2005;106:335 – 42. Korecka L, Bilkova Z, Holeapek M, Kralovsky J, Benes M, Lenfeld J, et al. Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments. J Chromatogr B 2004;808:15 – 24. Krenková J, Foret F. Immobilized microfluidic enzymatic reactors. Electrophoresis 2004;25:3550 – 63. Kricka LJ, Wilding P. Microchip PCR. Anal Bioanal Chem 2003;377:820 – 5. Kulys J. The carbon paste electrode encrusted with a microreactor as glucose biosensor. Biosens Bioelectron 1999;14:473 – 9. Lagally ET, Mathies RA. Integrated genetic analysis microsystems. J Phys, D, Appl Phys 2004;37:R245 – 61. 55 Laurell T, Drott J, Rosengren L. Silicon-wafer integrated enzyme reactors. Biosens Bioelectron 1995;10:289 – 99. Laurell T, Rosengren L. A micromachined enzyme reactor in (110)oriented silicon. Sens Actuators, B, Chem 1994;19:614 – 7. Lee TMH, Hsing I-M, Lao AIK, Carles MC. A miniaturized DNA amplifier: its application in traditional Chinese medicine. Anal Chem 2000;72:4242 – 7. Lendl B, Schindler R, Frank J, Kellner R. Fourier transform infrared detection in miniaturized total analysis systems for sucrose analysis. Anal Chem 1997;69:2877 – 81. L’Hostis E, Michel PE, Fiaccabrino GC, Strike DJ, de Rooij NF, Koudelka-Hep M. Microreactor and electrochemical detectors fabricated using Si and EPON SU-8. Sens Actuators, B, Chem 2000;64:156 – 62. Licklider L, Kuhr WG. Optimization of online peptide-mapping by capillary zone electrophoresis. Anal Chem 1994;66:4400 – 7. Licklider L, Kuhr WG. Characterization of reaction dynamics in a trypsin-modified capillary microreactor. Anal Chem 1998;70: 1902 – 8. Licklider L, Kuhr WG, Lacey MP, Keough T, Purdon MP, Takigiku R. Online microreactors/capillary electrophoresis/mass-spectrometry for the analysis of proteins and peptides. Anal Chem 1995;67:4170 – 7. Lilly MD, Hornby WE, Crook EM. Kinetics of carboxymethylcellulose-ficin in packed beds. Biochem J 1966;100:718 – 23. Liu DJ, Perdue RK, Sun L, Crooks RM. Immobilization of DNA onto poly(dimethylsiloxane) surfaces and application to a microelectrochemical enzyme-amplified DNA hybridization assay. Langmuir 2004;20:5905 – 10. Lv Y, Zhang Z, Chen F. Chemiluminescence microfluidic system sensor on a chip for determination of glucose in human serum with immobilized reagents. Talanta 2003;59:571 – 6. Ma L, Gong X, Yeung ES. Combinatorial screening of enzyme activity by using multiplexed capillary electrophoresis. Anal Chem 2000;72:3383 – 7. Madamwar DB, Bhatt JP, Ray RM, Srivastava RC. Activation and stabilization of invertase entrapped into reversed micelles of sodium lauryl sulfate and sodium tauroglycocholate in organic solvents. Enzyme Microb Technol 1988;10:302 – 5. Mao HB, Yang TL, Cremer PS. Design and characterization of immobilized enzymes in microfluidic systems. Anal Chem 2002;74:379 – 85. Maruyama T, Uchida J, Ohkawa T, Futami T, Katayama K, Nishizawa K, et al. Enzymatic degradation of p-chlorophenol in a two-phase flow microchannel system. Lab Chip 2003;3:308 – 12. Miyazaki M, Maeda H. Microreactor and method for producing the same. Patent No. JP2004267097;2004a. Miyazaki M, Maeda H. Microreactor with enzyme reversibly bonded therein and its production method. Patent No. JP2004160273; 2004b. Miyazaki M, Kaneno J, Kohama R, Uehara R, Kanno K, Fujii M, et al. Preparation of functionalized nanostructures on microchannel surface and their use for enzyme microreactors. Chem Eng J 2004;101:277 – 84. Moore BD, Stevenson L, Watt A, Flitsch S, Turner NJ, Cassidy C, et al. Rapid and ultra-sensitive determination of enzyme activities using surface-enhanced resonance Raman scattering. Nat Biotechnol 2004;22:1133 – 8. Moorthy J, Mensing GA, Kim D, Mohanty S, Eddington DT, Tepp WH, et al. Microfluidic tectonics platform: a colorimetric, disposable botulinum toxin enzyme-linked immunosorbent assay system. Electrophoresis 2004;25:1705 – 13. 56 P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 Müller G, Petry S. Lipases and phospholipases in drug development: from biochemistry to molecular pharmacology. Weinheim7 WileyVCH; 2004. Murakami Y, Takeuchi T, Yokoyama K, Tamiya E, Karube I, Suda M. Integration of enzyme-immobilized column with electrochemical flow cell using micromachining techniques for a glucose detection system. Anal Chem 1993;65:2731 – 5. Nagai H, Murakami Y, Yokoyama K, Tamiya E. High-throughput PCR in silicon based microchamber array. Biosens Bioelectron 2001;16:1015 – 9. Nakamura H, Li X, Wang H, Uehara M, Miyazaki M, Shimizu H, et al. A simple method of self assembled nano-particles deposition on the micro-capillary inner walls and the reactor application for photo-catalytic and enzyme reactions. Chem Eng J 2004;101: 261 – 8. Nashabeh W, Rassi ZE. Enzymophoresis of nucleic acids by tandem capillary enzyme reactor-capillary zone electrophoresis. J Chromatogr A 1992;596:251 – 64. Niwa O, Kurita R, Horiuchi T, Torimitsu K. Continuous monitoring of l-glutamate released from cultured rat nerve cells with a microfabricated on-line sensor at a slow flow rate. Electroanalysis 1999;11:356 – 61. Nomura A, Shin S, Mehdi OO, Kauffmann JM. Preparation, characterization, and application of an enzyme-immobilized magnetic microreactor for flow injection analysis. Anal Chem 2004;76: 5498 – 502. Oberholzer T, Meyer E, Amato I, Lustig A, Monnard P-A. Enzymatic reactions inside liposomes using the detergent-induced liposome loading method. Biochim Biophys Acta 1999;1416:57 – 68. Palm AK, Novotny MV. Analytical characterization of a facile porous polymer monolithic trypsin microreactor enabling peptide mass mapping using mass spectrometry. Rapid Commun Mass Spectrom 2004;18:1374 – 82. Park CB, Clark DS. Sol–gel encapsulated enzyme arrays for highthroughput screening of biocatalytic activity. Biotechnol Bioeng 2002;78:229 – 35. Park SS, Joo HS, Cho SI, Kim MS, Kim YK, Kim BG. Multi-step reactions on microchip platform using nitrocellulose membrane reactor. Biotechnol Bioprocess Eng 2003;8:257 – 62. Peterson DS. Solid supports for micro analytical systems Solid supports for micro analytical systems. Lab Chip 2005;5:132 – 9. Peterson DS, Rohr T, Svec F, Fréchet JMJ. High-throughput peptide mass mapping using a microdevice containing trypsin immobilized on a porous polymer monolith coupled to MALDI TOF and ESI TOF mass spectrometers. J Proteome Res 2002a;1:563 – 8. Peterson DS, Rohr T, Svec F, Fréchet JMJ. Enzymatic microreactoron-a-chip: protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices. Anal Chem 2002b;74:4081 – 8. Peterson DS, Rohr T, Svec F, Fréchet JMJ. Dual-function microanalytical device by in situ photolithographic grafting of porous polymer monolith: integrating solid-phase extraction and enzymatic digestion for peptide mass mapping. Anal Chem 2003;75:5328 – 35. Pfohl T, Mugele F, Seemann R, Herminghaus S. Trends in microfluidics with complex fluids. Chemphyschem 2003;4:1291 – 8. Pijanowska DG, Baraniecka A, Wiater R, Ginalska G, oobarzewski J, Torbicz W. The pH-detection of triglycerides. Sens Actuators, B, Chem 2001;78:263 – 6. Qu HY, Wang HT, Huang Y, Zhong W, Lu HJ, Kong JL, et al. Stable microstructured network for protein patterning on a plastic micro- fluidic channel: strategy and characterization of on-chip enzyme microreactors. Anal Chem 2004;76:6426 – 33. Regnier FE, Patterson DH, Harmon BJ. Electrophoretically-mediated microanalysis (EMMA). Trends Anal Chem 1995;14: 177 – 181. Rondelez Y, Tresset G, Tabata KV, Arata H, Fujita H, Takeuchi Sh, et al. Highly coupled ATP synthesis by F-1-ATPase single molecules. Nat Biotechnol:20 – 2. Samskog J, Bylund D, Jacobsson SP, Markides KE. Miniaturized online proteolysis-capillary liquid chromatographymass spectrometry for peptide mapping of lactate dehydrogenase. J Chromatogr A 2003;998:83 – 91. Sanders GHW, Manz A. Chip-based microsystems for genomic and proteomic analysis. Trends Anal Chem 2000;19:364 – 78. Schneegass I, Brautigam R, Kohler JM. Miniaturized flow-through PCR with different template types in a silicon chip. Lab Chip 2001;1:42 – 9. Schneegah I, Köhler JM. Flow-through polymerase chain reactions in chip thermocyclers. Rev Mol Biotechnol 2001;82:101 – 21. Schwarz MA, Hauser PC. Recent developments in detection methods for microfabricated analytical devices. Lab Chip 2001;1:1 – 6. Seong GH, Heo J, Crooks RM. Measurement of enzyme kinetics using a continuous-flow microfluidic system. Anal Chem 2003;75:3161 – 7. Srinivasan A, Bach H, Sherman DH, Dordick JS. Bacterial P450catalyzed polyketide hydroxylation on a microfluidic platform. Biotechnol Bioeng 2004;88:528 – 35. Strike DJ, Fiaccabrino G-C, Koudelka-Hep M, de Rooij NF. Enzymatic microreactor using Si, glass and EPON SU-8. Biomed Microdev 2000;2:175 – 8. Van Dyck S, Kaale E, Nováková S, Glatz Z, Hoogmartens J, Van Schepdael A. Advances in capillary electrophoretically mediated microanalysis. Electrophoresis 2003;24:3868 – 78. Verpoorte E. Beads and chips: new recipes for analysis. Lab Chip 2003a;3:60N – 8N. Verpoorte E. Chip vision — optics for microchips. Lab Chip 2003b;3:42N – 52N. Vilkner T, Janasek D, Manz A. Micro total analysis systems Recent developments. Anal Chem 2004;76:3373 – 86. Walde P, Ichikawa S. Enzymes inside lipid vesicles: preparation, reactivity and applications. Biomol Eng 2001;18:143 – 77. Washizu M, Yamamoto T, Kurosawa O, Suzuki S, Shimamoto N. Molecular surgery of DNA using enzyme-immobilized particles. Trans IEE Jpn 1996;116-E:196 – 202. Watts P, Wiles C, Haswell SJ, Pombo-Villar E, Styring P. The synthesis of peptides using micro reactors. Chem Commun 2001:990 – 1. Wilhelm T, Wittstock G. Generation of periodic enzyme patterns by soft lithography and activity imaging by scanning electrochemical microscopy. Langmuir 2002;18:9485 – 93. Xie B, Danielsson B, Norberg P, Winquist F, Lundström I. Development of a thermal micro-biosensor fabricated on a silicon chip. Sens Actuators, B, Chem 1992;6:127 – 30. Xie Sh, Svec F, Fréchet JMJ. Design of reactive porous polymer supports for high throughput bioreactors: poly(2-vinyl-4,4dimethylazlactone-co-acrylamide-co-ethyl dimethacrylate) monoliths. Biotechnol Bioeng 1999;62:30 – 5. Xu Z-R, Fang Z-L. Composite poly(dimethylsiloxane)/glass microfluidic system with an immobilized enzymatic particle-bed reactor and sequential sample injection for chemiluminescence determinations. Anal Chim Acta 2004;507:129 – 35. P.L. Urban et al. / Biotechnology Advances 24 (2006) 42–57 Yadavalli VK, Koh W-G, Lazur GJ, Pishko MV. Microfabricated protein-containing poly(ethylene glycol) hydrogel arrays for biosensing. Sens Actuators, B, Chem 2004;97:290 – 7. Yakovleva J, Davidsson R, Lobanova A, Bengtsson M, Eremin S, Laurell T, et al. Microfluidic enzyme immunoassay using silicon microchip with immobilized antibodies and chemiluminescence detection. Anal Chem 2002;74:2994 – 3004. Ye M, Hu Sh, Schoenherr RM, Dovichi NJ. On-line protein digestion and peptide mapping by capillary electrophoresis with post-column labeling for laser-induced fluorescence detection. Electrophoresis 2004;25:1319 – 26. 57 Zhan W, Seong GH, Crooks RM. Hydrogel-based microreactors as a functional component of microfluidic systems. Anal Chem 2002;74:4647 – 52. Zhang N, Yeung ES. On-line coupling of polymerase chain reaction and capillary electrophoresis for automatic DNA typing and HIV1 diagnosis. J Chromatogr B 1998;714:3 – 11. Zhang YY, Tadigadapa S. Calorimetric biosensors with integrated microfluidic channels. Biosens Bioelectron 2004;19:1743 – 73. Zhao CA, Wittstock G. Scanning electrochemical microscopy of quinoprotein glucose dehydrogenase. Anal Chem 2004;76: 3145 – 54.