Gold nanoparticle assembly microfluidic reactor for efficient on-line proteolysis

Mol Cell Proteomics. 2007 Aug;6(8):1428-36. doi: 10.1074/mcp.T600055-MCP200. Epub 2007 May 22.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allyl Compounds / chemistry*
  • Amino Acid Sequence
  • Animals
  • Biosensing Techniques*
  • Cell Line
  • Gold / chemistry*
  • Macrophages / chemistry*
  • Metal Nanoparticles / chemistry*
  • Microfluidic Analytical Techniques / methods*
  • Molecular Sequence Data
  • Polyethylene Terephthalates
  • Proteome / analysis*
  • Quaternary Ammonium Compounds / chemistry*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Trypsin / metabolism

Substances

  • Allyl Compounds
  • Polyethylene Terephthalates
  • Proteome
  • Quaternary Ammonium Compounds
  • Gold
  • diallyldimethylammonium chloride
  • Trypsin