Microreactor enzyme

Therefore, minimal sample handling is advisable. In order to overcome the problem of limited sample loading, Guzman conceived and demonstrated the concept of on-line preconcentration with CE using a cartridge containing a bed of adsorptive phase (5). In the present work we describe the use of nonspecific on-line preconcentration-CE, on-line immunoaffinity-CE (lA-CE) and on-line microreactor enzyme digestion-CE for the analyses of proteins. 

Figure 3 Schematic of a microreactor enzyme digestion chamber couples to CE.

PMMA enzymatic microreactor enzyme and substrate co-flowed through T-channel mixing by staggered herring- bone micro-mixer (SHM) Integrated with filtration unit using gaskets made from PDMS... 

The resulting enzyme-containing microcapsules (which can contain different enzymes in different capsules, as was the case here) were then embedded within a Ca-alginate bead, designated a capsules-in-bead structured microreactor (Scheme 5.7). 

The three different enzymes used in combination in this system were FateDH, FaldDH, andADH. FateDH catalyzes the initial reduction of C02 to formate, FaldDH the reduction of formate to formaldehyde, and ADH the reduction of formaldehyde to methanol. Interestingly, the enzymes when immobilized were more active than a combination of the free enzymes, which is claimed to be due to a reduction of spatial interference among the different enzymes. Moreover, due to the immobilization of enzymes within the microreactor, the intermediate species have significantly reduced distances to travel between active sites [21, 22]. 

Scheme 5.7 Encapsulation of enzyme microcapsules into a gel-like structure (host gel bead) resulting in a capsules-in-bead microreactor. Reproduced from [20] by permission of The Royal Society of Chemistry.

As mentioned above, in order to extend the potentialities of the luminescence-based optical fibre biosensors to other analytes, auxiliary enzymes can be used. The classical approaches consist either of the coimmobilization of all the necessary enzymes on the same membrane or of the use of microreactors including immobilized auxiliary enzymes and... 

Water in oil microemulsions with reverse micelles provide an interesting alternative to normal organic solvents in enzyme catalysis with hydrophobic substrates. Reverse micelles are useful microreactors because they can host proteins like enzymes. Catalytic reactions with water insoluble substrates can occur at the large internal water-oil interface inside the microemulsion. The activity and stability of biomolecules can be controlled, mainly by the concentration of water in these media. With the exact knowledge of the phase behaviom" and the corresponding activity of enzymes the application of these media can lead to favomable effects compared to aqueous systems, like hyperactivity or increased stability of the enzymes. 

A further improvement of the multiphase reactor concept using lipase for enantioselective transformation has been recently reported, that is, an emulsion enzyme membrane reactor. Here, the organic/water interface within the pores at the enzyme level is achieved by stable oil-in-water emulsion, prepared by membrane emulsification. In this way, each pore forms a microreactor containing immobilized... 

For periodic reuse of the enzymes a new project was developed including a microreactor incorporated into the FIA system. The micro-reactor shown in Fig. 2, made of acrylic acid and with a 0.91-mL void volume, and length-to-diameter ratio of 3 1, was packed with AOD immobilized on glass beads. The beads were retained in the microreactor with a 110-mesh nylon screen and two rubber O-rings with an 11.4-mm external diameter. The lids were attached to the microreactor with four stainless steel screws. 

Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon. The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of >90% were observed. 

Index Entries Microscale bioreactor polydimethylsiloxane microreactor immobilized enzymes urease enzyme silicon wafer. 

In this article, we report on the fabrication and performance of microreactors constructed of silicon and polydimethylsiloxane (PDMS). The resulting structures contain immobilized enzymes for converting biochemical substrates to useful products or for breaking down organics into waste streams. 

Urease (EC 3.5.1.5 Type IX, Sigma-Aldrich from Jack Beans) was used throughout the experiments. Before immobilizing urease onto the microreactor systems, the enzyme was evaluated for activity in the chosen buffer system (Tris[hydroxymethyl]aminomethane [THAM]). Free enzyme tests of the urease showed an approximate activity of 44,800 U/g of solid. 

Continuous studies were performed in specially prepared microreactors molded from PDMS, designated PDMS (Sylgard 184 silicone elastomer Dow Corning) poured onto silicon wafer molds. The microreactor molds were prepared using 4-in. silicon wafers of Type P, crystal orientation of , resistivity of 1 to 2 Q, and thickness of 457-575 pm from Silicon Quest (Santa Clara, C A). After preparation, mixtures of urease enzyme and PDMS (designated PDMS-E) were poured onto the microreactor mold and allowed to cure at ambient conditions. 

A negative photoresist, SU-8 (Microchem), was used in the microreactor mold process for preparing the PDSM-E microreactors. When exposed to ultraviolet light, material may be removed via a wet etching process leaving high-definition features in micrometer dimensions. Additionally, a microreactor has been constructed in silicon onto which layer-bylayer self-assembled polyelectrolytes and enzymes are deposited. This system is being used for comparison with the PDMS-E system performance. 

The combination of PDMS and urease enzyme to form a microreactor from the resulting "bioplastic" material (PDMS-E) has been reported previously (7). When enzyme concentrations were maintained at 2.5% (w/w) or less, the resulting microreactor cured with good structural integrity and high definition (e.g., well-formed microchannels and >90% retention of triangular transverse packing features in the microchannels). 

For enzyme attachment to the silicon microreactor tested, a layer-by-layer technique was employed to build a multilayer system of polyions and enzyme. Deposition of multilayers was accomplished by alternating positively and negatively charged layers of polydimethyldiallyl ammonium chloride (PDDA) and polystyrene sulfonate (PSS), respectively, to which was attached urease enzyme. After depositing in succession three layers of PDDA, PSS, and PDDA, three layers of urease enzyme were alternately deposited with three layers of PDDA. The resulting architecture is described as follows ... 

For the urease enzyme system, a reactant solution ofO.lmol/Lofurea was fed to the microreactors by Cole Parmer Series 74900 Syringe pumps. 

The PDMS-E described in the batch studies was used to mold reactors. These microreactors were fed the same 0.1 M urea solution as used in batch experiments. Reactors were operated for approx 1 hbefore acquiring operational data to reduce the effects of any loosely bound enzymes that may wash out from the surfaces of the microchannel walls. 

In order to increase the efficiency of biocatalytic transformations conducted under continuous flow conditions, Honda et al. (2006, 2007) reported an integrated microfluidic system, consisting of an immobilized enzymatic microreactor and an in-line liquid-liquid extraction device, capable of achieving the optical resolution of racemic amino acids under continuous flow whilst enabling efficient recycle of the enzyme. As Scheme 42 illustrates, the first step of the optical resolution was an enzyme-catalyzed enantioselective hydrolysis of a racemic mixture of acetyl-D,L-phenylalanine to afford L-phenylalanine 157 (99.2-99.9% ee) and unreacted acetyl-D-phenylalanine 158. Acidification of the reaction products, prior to the addition of EtOAc, enabled efficient continuous extraction of L-phenylalanine 157 into the aqueous stream, whilst acetyl-D-phenylalanine 158 remained in the organic fraction (84—92% efficiency). Employing the optimal reaction conditions of 0.5 gl min 1 for the enzymatic reaction and 2.0 gl min-1 for the liquid-liquid extraction, the authors were able to resolve 240 nmol h-1 of the racemate. 

Employing a multichannel PDMS microreactor [350 gm (wide) x 250 gm (deep) x 6.4 mm (long)], in which the thermophilic enzyme (3-glycosi-dase was immobilized, Thomsen et al. (2007) evaluated the hydrolysis of 2-nitrophenyl-p-D-galactopyranoside. Heating the reactor to 80 °C, the authors were able to continuously hydrolyze 2-nitrophenyl-p-D-galactopyranoside and monitored the reaction efficiency via generation of 2-nitrophenol 97. 

Liposomes can even be used as microreactors. Until recently, the utility of this technique was limited by the fact that they could be used for only a relatively short time because of depletion of the reaction mixture. It has now been shown that liposomes of l-palmitoyl-2-oleoyl-sn-glycero-3-phosphochoHne (POPC) can be used as a semipermeable microreactor after treatment with sodium cholate. It has been demonstrated that this allows a biochemical reaction to take place inside the liposomes but not in the external medium. Such cholate-induced POPC bilayers can also be used to insert enzymes [91]. 

More recently, microreactor technology has entered the field of biocatalysis enzymes are used for synthesis rather than for diagnostics. The concept behind the use of biocatalytic microreactor systems is in fact twofold. First, a miniaturized reactor allows an efficient use of small amounts of enzyme, when enzyme kinetics determination is involved. Second, the classical advantages of microreactors in synthesis, namely, better control over heat- and mass-transfer... 

Besides the benefits of scale reduction and trypsin immobilization, microsystem technology has other advantages to offer. For example, Ekstrom et al. [345] described a device that integrated an enzyme microreactor with a sample pretreatment robot and... 

The examples of bioorganic chemistry in the previous paragraph are all concerned with the known biocatalytic assays, in which the effects of miniaturization on the efficiency of the analytical method were investigated. Recently, a new development has started in which the biocatalytic process itself has become the center of attention. Biocatalysis in microreactors, as described in here, deal with the investigation of the use of enzymes for the production of molecules. Two different approaches can be identified. In one line of investigation, the miniaturized reaction environment is used to screen the efficiency of an enzyme. In this case, only small amounts of... 

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