Assembly-controlled biocompatible interface on a microchip: strategy to highly efficient proteolysis.
A biocompatible interface was constructed on a microchip by using the layer-by-layer (LBL) assembly of charged polysaccharides incorporating proteases for highly efficient proteolysis. The controlled assembly of natural polyelectrolytes and the enzyme-adsorption step were monitored by using a quartz-crystal microbalance and atomic force microscopy (AFM). Such a multilayer-assembled membrane provides a biocompatible interconnected network with high enzyme-loading capacity. The maximum digestion rate of the adsorbed trypsin in a microchannel was significantly accelerated to 1600 mM min(-1) microg(-1), compared with the tryptic digestion in solution. Based on the Langmuir isotherm model, the thermodynamic constant of adsorption K was calculated to be 1.6 x 10(5) M(-1) and the maximum adsorption loading Gammamax was 3.6 x 10(-6) mol m(-2), 30 times more than a monolayer of trypsin on the native surface. The tunable interface containing trypsin was employed to construct a microchip reactor for digestion of femtomoles of proteins and the produced peptides were analyzed by MALDI-TOF mass spectroscopy. The efficient on-chip proteolysis was obtained within a few seconds, and the identification of biological samples was feasible.
Liu
Yun
Y
Department of Chemistry, Institute of Biomedical Sciences, Fudan University, Shanghai 200433, P. R. China.
Zhong
Wei
W
Meng
Sheng
S
Kong
Jilie
J
Lu
Haojie
H
Yang
Pengyuan
P
Girault
Hubert H
HH
Liu
Baohong
B
D001672:Biocompatible Materials / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods; D002364:Caseins / Q000032:analysis / Q000378:metabolism; D048271:Chitosan / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D022362:Hepatitis A Vaccines / Q000032:analysis / Q000378:metabolism; D006820:Hyaluronic Acid / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D046210:Microfluidic Analytical Techniques; D018625:Microscopy, Atomic Force; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
D001672:Biocompatible Materials; D002364:Caseins; D004800:Enzymes, Immobilized; D022362:Hepatitis A Vaccines; D010455:Peptides; D006820:Hyaluronic Acid; D048271:Chitosan; D014357:Trypsin