TY - JOUR
T1 - Enzymatic microreactor-on-a-chip
T2 - Protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices
AU - Peterson, Dominic S.
AU - Rohr, Thomas
AU - Svec, Frantisek
AU - Fréchet, Jean M.J.
PY - 2002/8/15
Y1 - 2002/8/15
N2 - 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-α-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 μL/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%.
AB - 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-α-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 μL/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%.
UR - http://www.scopus.com/inward/record.url?scp=0037102287&partnerID=8YFLogxK
U2 - 10.1021/ac020180q
DO - 10.1021/ac020180q
M3 - Article
C2 - 12199578
AN - SCOPUS:0037102287
SN - 0003-2700
VL - 74
SP - 4081
EP - 4088
JO - ANALYTICAL CHEMISTRY
JF - ANALYTICAL CHEMISTRY
IS - 16
ER -