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%.

Matrix assisted laser desorption/ionization (MALDI) is a soft ionization mass spectrometric method that has become a preeminent technique in the analysis of a wide variety of compounds including polymers and proteins. The main drawback of MALDI is that it is difficult to analyze low molecular weight compounds (<1,000 m/z) because the matrix that allows MALDI to work interferes in this mass range. In recent years there has been considerable interest in developing laser desorption/ionization (LDI) techniques for the analysis of small molecules. This review examines the approaches to matrix-free LDI mass spectrometry including desorption/ionization on silicon (DIOS), sol-gels, and carbon-based microstructures. For the purposes of this review matrix-free methods are defined as those that do not require matrix to be mixed with the analyte and therefore does not require co-crystallization. The review will also examine mechanisms of ionization and applications of matrix-free LDI-MS.

Microfluidic devices with a dual function containing both a solid-phase extractor and an enzymatic microreactor have been prepared, and their operation has been demonstrated. The devices were fabricated from a 25-mm-long porous poly(butyl methacrylate-co-ethylene dimethacrylate) monolith prepared within a 50-microm-i.d. capillary. This capillary with a pulled 9-12-microm needle tip was used as a nanoelectrospray emitter coupling the device to a mass spectrometer. Photografting with irradiation through a mask was then used to selectively functionalize a 20-mm-long portion of the monolith, introducing reactive poly(2-vinyl-4,4-dimethylazlactone) chains to enable the subsequent attachment of trypsin, thereby creating an enzymatic microreactor with high proteolytic activity. The other 5 mm of unmodified hydrophobic monolith served as micro solid-phase extractor (microSPE). The dual-function devices were used in two different flow directions; concentration of myoglobin that was absorbed from its dilute solution, followed by elution and digestion or digestion, followed by concentration. Operations in both directions afforded equal sequence coverage. Different volumes of myoglobin solution ranging from 2 to 20 microL were loaded on the device. Very high sequence coverages of almost 80% were achieved for the highest loading. Despite the very short length of the extractor unit, the device operated in the digest-solid-phase extraction direction also enabled the separation of peaks that mostly contained undigested protein and peptides.