Multilayer-assembled microchip for enzyme immobilization as reactor toward low-level protein identification.
A microchip reactor has been developed on the basis of a layer-by-layer approach for fast and sensitive digestion of proteins. The resulting peptide analysis has been carried out by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Natural polysaccharides, positively charged chitosan (CS), and negatively charged hyaluronic acid (HA) were multilayer-assembled onto the surface of a poly(ethylene terephthalate) (PET) microfluidic chip to form a microstructured and biocompatible network for enzyme immobilization. The construction of CS/HA assembled multilayers on the PET substrate was characterized by AFM imaging, ATR-IR, and contact angle measurements. The controlled adsorption of trypsin in the multilayer membrane was monitored using a quartz crystal microbalance and an enzymatic activity assay. The maximum proteolytic velocity of the adsorbed trypsin was approximately 600 mM/min mug, thousands of times faster than that in solution. BSA, myoglobin, and cytochrome c were used as model substrates for the tryptic digestion. The standard proteins were identified at a low femtomole per analysis at a concentration of 0.5 ng/muL with the digestion time <5s. This simple technique may offer a potential solution for low-level protein analysis.
Liu
Yun
Y
Department of Chemistry, Institutes of Biomedical Sciences, Fudan University, Shanghai, People's Republic of China.
Lu
Haojie
H
Zhong
Wei
W
Song
Pengyu
P
Kong
Jilie
J
Yang
Pengyuan
P
Girault
Hubert H
HH
Liu
Baohong
B
D000327:Adsorption; D048271:Chitosan / Q000737:chemistry; D045304:Cytochromes c / Q000032:analysis*; D004800:Enzymes, Immobilized / Q000032:analysis*; D006820:Hyaluronic Acid / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D009211:Myoglobin / Q000032:analysis*; D011092:Polyethylene Glycols / Q000737:chemistry; D011093:Polyethylene Terephthalates; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013499:Surface Properties; D014357:Trypsin / Q000032:analysis*
D004800:Enzymes, Immobilized; D009211:Myoglobin; D011093:Polyethylene Terephthalates; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D006820:Hyaluronic Acid; D045304:Cytochromes c; D048271:Chitosan; D014357:Trypsin