Microreactor protein-immobilized

In another investigation, ELP[V5L2G3-90] with three lysines in the N-terminal region was immobihzed on a glass surface in a microreactor to enable temperature-controlled positioning of ELP fusion proteins. For this purpose, the glass surface was first functionalized with A -2-(aminoethyl)-3-aminopropyltrimethoxysilane, followed by glutaraldehyde treatment and reductive amination to immobilize the biopolymer on the surface (Fig. 17b) [132]. 

Peterson, D.S., Rohr, T., Svec, F., Frechet, J. M.J., Enzymatic microreactor-on-a-chip Protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices. Anal. Chem. 2002, 74(16), 4081M088. 

SAMs on microchannel walls have been studied for surface properties in microreactors,59 to control surface wetting,60 to create zones for specific immobilization of proteins and biomolecules,61 and to conduct catalytic reactions.62 And a pH sensing monolayer confined to a glass microchannel has been reported by our group.32... 

A silica sol-gel monohth containing zeolite nanoparticles has been reported [87]. This device had a high stuface area for the immobilized enzyme, allowing for a high load within the microreactor. A 0.5 pL volume containing 0.2 pg/pL of the proteins (cytochrome c and BSA) was digested within 5 s in the microreactor, as indicated by off-line MALDI-TOF (time-of-flight) MS. The microreactor could be used repeatedly and the enzyme remained active for more than a month when it was stored at temperatures below 4 °C. 

In addition to serving as the stationary phases, monolithic materials are also finding numerous other applications in the microfluidic world. Their use in on-chip solid-phase extraction and preconcentration,as supports for immobilization of enzymes to form enzymatic microreactors for protein mapping,static mixers,and valves represent just a few examples of modules in the microfluidic toolbox and further growth is inevitable. 

One solution to the challenges of mixing proteins in microfluidic systems is to immobilize proteins in microreactors [5]. These systems typically consist of chambers of enzymes immobilized on beads, micropillars [6], or porous polymer monoliths [7] (Fig. 2a and b). Such systems have large surface area-to-volume ratios, which minimize diffusion time for reactions with solution-phase reagents. Microreactors can be used either for the conversion of an analyte to another form that is more easily detected or for direct studies of the properties of enzymes and substrates. One of the most common uses is for the digestion of proteins for proteome profiling, but such systems can also be used for the removal of amino acid residues from peptides or proteins or for enzyme kinetic studies. 

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]. 

Monolithic capillary microreactor Trypsin Covalent linking Hybrid silica monolith-based immobilized trypsin microreactor for on-line protein digestion of 4-protein mixture BSA, myoglobin, P-globulin, and ribonuclease Sequence coverage were 8,26,10, and 54%, respectively Proteins extracted from E. coli digested and lOlproteins identified. [131]... 

Monolithic capillary microreactor Trypsin Adsorption Trypsin immobilized on matrix-HILIC by dynamic immobilization method high hydrolytic activity of BAPNA high enzymatic activity of standard proteins BSA, Cyt-C, and myoglobin sequence coverage of myoglobin and Cyt-C >50% [136]... 

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. 

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... 

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