Gold nanoparticle assembly microfluidic reactor for efficient on-line proteolysis.
A microchip reactor coated with a gold nanoparticle network entrapping trypsin was designed for the efficient on-line proteolysis of low level proteins and complex extracts originating from mouse macrophages. The nanostructured surface coating was assembled via a layer-by-layer electrostatic binding of poly(diallyldimethylammonium chloride) and gold nanoparticles. The assembly process was monitored by UV-visible spectroscopy, atomic force microscopy, and quartz crystal microbalance. The controlled adsorption of trypsin was theoretically studied on the basis of the Langmuir isotherm model, and the fitted Gamma(max) and K values were estimated to be 1.2 x 10(-7) mol/m(2) and 4.1 x 10(5) M(-1), respectively. An enzymatic kinetics assay confirmed that trypsin, which was entrapped in the biocompatible gold nanoparticle network with a high loading capacity, preserved its bioactivity. The maximum proteolytic rate of the adsorbed trypsin was 400 mM/(min.microg). Trace amounts of proteins down to femtomole per analysis were digested using the microchip reactor, and the resulting tryptic products were identified by MALDI-TOF MS/MS. The protein mixtures extracted from the mouse macrophages were efficiently identified by on-line digestion and LC-ESI-MS/MS analysis.
Liu
Yun
Y
Department of Chemistry and Institute of Biomedical Sciences, Fudan University, Shanghai 200433, China.
Xue
Yan
Y
Ji
Ji
J
Chen
Xian
X
Kong
Jilie
J
Yang
Pengyuan
P
Girault
Hubert H
HH
Liu
Baohong
B
D000498:Allyl Compounds / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques; D002460:Cell Line; D006046:Gold / Q000737:chemistry*; D008264:Macrophages / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D011093:Polyethylene Terephthalates; D020543:Proteome / Q000032:analysis*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D000498:Allyl Compounds; D011093:Polyethylene Terephthalates; D020543:Proteome; D000644:Quaternary Ammonium Compounds; D006046:Gold; C508190:diallyldimethylammonium chloride; D014357:Trypsin