The use of enzymes for cleavage, synthesis or chemical modification represents one of the most common processes used in biochemical and molecular biology laboratories. The continuing progress in medical research, genomics, proteomics, and related emerging biotechnology fields leads to exponential growth of the applications of enzymes and the development of modified or new enzymes with specific activities. Concurrently, new technologies are being developed to improve reaction rates and specificity or perform the reaction in a specific environment. Besides large-scale industrial applications, where typically a large processing capacity is required, there are other, much lower-scale applications, benefiting form the new developments in enzymology. One such technology is microfluidics with the potential to revolutionize analytical instrumentation for the analyses of very small sample amounts, single cells or even subcellular assemblies. This article aims at reviewing the current status of the development of the immobilized microfluidic enzymatic reactors (IMERs) technology.

Capillary enzymatic microreactors containing trypsin and endoproteinase LysC immobilized on a porous polymer monolith have been prepared and used for the characterization and identification of proteins such as cytochrome c, bovine serum albumin, and high-molecular weight human immunoglobulin G. The hydrophilicity of diol functionalities originating from the hydrolyzed poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith was not sufficient to avoid adsorption of hydrophobic albumin in a highly aqueous mobile phase. Therefore, this monolith was first hydrophilized via photografting of poly(ethylene glycol) methacrylate followed by photografting of a 4-vinyl-2,2-dimethylazlactone to provide the pore surface with reactive functionalities required for immobilization. This new approach reduced the undesired nonspecific adsorption of proteins and peptides and facilitated control of both the enzyme immobilization and protein digestion processes. The enzymatic reactors were coupled off-line with MALDI/TOF MS and/or on-line with ESI/TOF MS. Experimental conditions for digestion were optimized using cytochrome c and bovine serum albumin as model proteins. The optimized reactors were then integrated into a multidimensional system comprised of a monolithic capillary enzyme reactor, an in-line nanoLC separation of peptides using a poly(lauryl methacrylate-co-ethylene dimethacrylate) monolithic column, and ESI/TOF MS. With the use of this system, immunoglobulin G was digested at room temperature in 6 min to an extent similar to that achieved with soluble enzyme at 37 degrees C after 24 h.

New monolithic capillary columns with embedded commercial hydroxyapatite nanoparticles have been developed and used for protein separation and selective enrichment of phosphopeptides. The rod-shaped hydroxyapatite nanoparticles were incorporated into the poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith by simply admixing them in the polymerization mixture followed by in situ polymerization. The effect of percentages of monomers and hydroxyapatite nanoparticles in the polymerization mixture on the performance of the monolithic column was explored in detail. We found that the loading capacity of the monolith is on par with other hydroxyapatite separation media. However, the speed at which these columns can be used is higher due to the fast mass transport. The function of the monolithic columns was demonstrated with the separations of a model mixture of proteins including ovalbumin, myoglobin, lysozyme, and cytochrome c as well as a monoclonal antibody and its aggregates with protein A. Selective enrichment and MALDI/MS characterization of phosphopeptides fished-out from complex peptide mixtures of ovalbumin, α-casein, and β-casein digests were also achieved using the hydroxyapatite monolith.