Enzymatic microreactors have been prepared in capillaries and on microfluidic chips by immobilizing trypsin on porous polymer monoliths consisting of 2-vinyl-4,4-dimethylazlactone, ethylene dimethacrylate, and acrylamide or 2-hydroxyethyl methacrylate. The azlactone functionalities react readily with amine and thiol groups of the enzyme to form stable covalent bonds. The optimized porous properties of the monoliths lead to very low back pressures enabling the use of simple mechanical pumping to carry out both the immobilization of the enzyme from its solution and the subsequent analyses of substrate solutions. The Michealis-Menten kinetic characteristics of the reactors were probed using a low molecular weight substrate: N-alpha-benzoyl-L-arginine ethyl ester. The effects of immobilization variables such as the concentration of trypsin in solution and percentage of azlactone functionalities in the monolith, as well as the effect of reaction time on the enzymatic activity, and of process variables such as substrate flow velocity and residence time in the reactor, were studied in detail. The proteolytic activity of the enzymatic microreactor on chip was demonstrated at different flow rates with the cleavage of fluorescently labeled casein used as a substrate. The excellent performance of the monolithic microreactor was also demonstrated with the digestion of myoglobin at the fast flow rate of 0.5 microL/min, which affords a residence time of only 11.7 s. The digest was then characterized using MALDI-TOF MS, and 102 out of 153 possible peptide fragments were identified giving a sequence coverage of 67%.

The photografting of porous three-dimensional materials has been achieved using a benzophenone-initiated surface photopolymerization within the pores of a macroporous polymer monolith contained in a fused silica capillary. Despite the relatively high thickness (100 µm or more) of the layer of material involved, the photografting process occurs efficiently throughout its cross section as confirmed by electron probe microanalysis. In addition, the use of photomasks during grafting enables the precise placement of specific functionalities in selected and predetermined areas of a single monolith for use in a variety of applications ranging from supported catalysis to microfluidics. For example, we have demonstrated the fast and selective incorporation of chains of poly(2-acrylamido-2-methyl-1-propanesulfonic acid) into the irradiated areas of pores of a 100 µm thick monolith and monitored the extent of grafting through measurements of the electroosmotic flow afforded by the newly introduced ionized functionalities. Grafting of the porous polymer with 4,4-dimethyl-2-vinylazlactone was also successful and could be monitored visually by fluorescence measurements following fluorescent labeling of the grafted chains with Rhodamine 6G.