Enzyme-immobilized capillary microreactor

Immobilization of Enzyme in Capillary Microreactor IMER systems in capillary microreactors are now routinely being used in protein digestion for peptide mapping and proteomics. Similar to microfluidic charmels on chips, enzyme immobilization in capillary can be classified into two categories based on the immobilization location. 

Immobilization of Enzyme in Capillary Microreactor by Entrapment Entrapment technique involves the entrapment of enzymes in gel matrix. The enzyme is mixed with gel formation ingredients and upon gel formation, the enzyme remains trapped in the matrix. Another form of entrapment is the formation of membrane around the droplet of enzyme, which is typically in solution. Immobilization by entrapment differs from adsorption and covalent bonding in that enzymes are free in solution but restricted in movement by the lattice structure of a gel. The membrane must be permeable to diffusion of substrate and product molecules... 

Smirnova et al. demonstrated the determination of the insecticide, carbaryl, using a two-chip system. The first chip (for the hydrolysis of carbaryl) had a simple Y-shaped channel while the second chip (for the diazo couphng reaction between hydrolyzed products and 2,4,6-trimethylaniline)—the extraction required special channel shapes with a partial surface— modification obtained by using capillary-restricted modification (CARM) (Figure 35.11). " Determination of carbaryl pesticide in water with sufficient sensitivity was carried out with an analysis time of 8 min. In a similar manner, Honda et al. developed a combination of a tube-type enzyme-immobilized microreactor and a microextractor with partial surface modification to produce optically pure amino acids. 

For both diastereomeric products. Enantiomeric excess of the residual substrate depended on the conversion. Capillary microreactors (CMRs) charged with the corresponding His -tagged enzyme bound to immobilized metal... 

To fulfill such requirements, attempts have been made in the past decade by researchers working on peptide mapping and proteomics through development of immobilized microfluidic enzymatic reactors. Microfluidic enzymatic microreactors are an alternative to in-solution method employing immobilization of proteases on microchaimels of chip-based reactors or surfaces of capillaries. The microreactors that enable proteolytic digestion by enzymes immobilized on solid supports are also referred to as immobilized enzyme reactors, IMERs. The great potential of IMERS for proteomic applications comprise rapid and enhance... 

Open tubular capillary microreactor Trypsin Entrapment Immobilized trypsin reactor prepared by enacapsulating trypsin in hydrogel. Enzymatic activity 700 times higher than enzyme in free solution. Enzymatic activity was evaluated using a-N-benzoyl-i-arginine ethyl bradyldnin ester and two peptides (bradykinin and [Tyr8]-) [107]... 

Another category of enzymatic transformations in multiphase systems is enzymes immobilized on the reactor wall as presented in Table 10.4. Enzymes are advantageously used in immobilized form because this strategy allows for increased volumetric productivity and improves stability. Continuous mode of operation is employed in these systems. The approaches commonly used for immobilization in conventional multiphase biocatalysis can also be employed in microreactors such as covalent methods, cross-linked enzyme aggregates (CLEA), and adsorption methods. The experimental setups can either be chip-type reactors with activated charmel surface walls where enzyme binds, or enzyme immobilized monolith reactors, where a support is packed inside a capillary tube. 

Biocatalytic reactions performed using immobilized enzyme microreactors under continuous flow mode have been found effective for hydrolysis reactions [121,158-161], with the enzyme either trapped in the matrix [159], covalently linked to modified surface wall [160,121], enzymes entrapped in hydrogels [162], or enzymes immobilized on monolith [179]. The experimental setup consists of either chip-type microreactors with activated chaimel walls where enzymes bind, enzymes that bind to beads, enzymes entrapped in the matrix, enzymes adsorbed in nanoporous materials, and most recently, nanosprings as supports for immobilized enzymes in chip-based reactors, or enzyme immobilized monolith reactors, where support is packed inside a capillary tube (Table 10.4). 

Literature on enzyme microreactors for chemical synthesis is scarce. There is abundant literature, however, on the use of enzymes in microsystems for purposes of DNA analysis, e.g. using the polymerase chain reaction discussed before or DNA restriction fragment analysis [81], for proteomics, such as tryptic digestion of proteins with the enzyme free in solution [82], with trypsin-coated beads trapped in a microreactor [83] or trypsin immobilized on a porous polymer monolith (in a fused-silica capillary) [84]. Also, enzyme microreaction devices have been used extensively for medical diagnostics or immunoassays, e.g. using porous silicon as a support for immobilized enzymes [85]. 

Zhao s group also integrated an immobilized enzyme microreactor in fused silica capillaries with a nanoelectrospray mass spectrometry for on-line digestion and fast peptide mass mapping [118]. Trypsin was immobilized onto the surface of the inner... 

Covalent linking of enzyme on fused silica capillaries was also demonstrated by Stigter [119]. Pepsin was covalently immobilized on dextran-modified capillaries as illustrated in Scheme 10.12. The applicability of pepsin-immobilized microreactor was tested with a number of proteins varying in molecular weight, isoelectric point, and sample composition. This open tubular microreactor demonstrated a flow-dependent digestion as represented by native hemoglobin. Complete digestion of... 

An integrated platform with the combination of protein and peptide separation was established online by Wang and group, by which proteins were first separated by capillary isoelectric focusing, online digested by a trypsin immobilized enzyme microreactor, trapped and desalted by two parallel columns, separated by nanoreversed phase and finally identified by MS (Figure 10.22)... 

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