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Molecular & Cellular Proteomics 6:1428-1436, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

Gold Nanoparticle Assembly Microfluidic Reactor for Efficient On-line Proteolysis^*,S

Yun Liu^,, Yan Xue^,, Ji Ji, Xian Chen^,¶, Jilie Kong, Pengyuan Yang, Hubert H. Girault^|| and Baohong Liu^,**

From the Department of Chemistry and Institute of Biomedical Sciences, Fudan University, Shanghai 200433, China, ^¶ Department of Biochemistry and Biophysics School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, and ^|| Laboratoire d'Electrochimie Physique et Analytique, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland

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 _max and K values were estimated to be 1.2 x 10^–7 mol/m² and 4.1 x 10⁵ 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·µg). 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.

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