Assembly-controlled biocompatible interface on a microchip: strategy to highly efficient proteolysis

Chemistry. 2006 Aug 25;12(25):6585-91. doi: 10.1002/chem.200501622.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biocompatible Materials / chemistry*
  • Biosensing Techniques / methods
  • Caseins / analysis
  • Caseins / metabolism
  • Chitosan / chemistry
  • Enzymes, Immobilized / chemistry*
  • Hepatitis A Vaccines / analysis
  • Hepatitis A Vaccines / metabolism
  • Hyaluronic Acid / chemistry
  • Hydrophobic and Hydrophilic Interactions
  • Kinetics
  • Microfluidic Analytical Techniques
  • Microscopy, Atomic Force
  • Peptides / analysis
  • Peptides / metabolism
  • Protein Array Analysis / methods*
  • Proteomics / methods
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Trypsin / chemistry*
  • Trypsin / metabolism

Substances

  • Biocompatible Materials
  • Caseins
  • Enzymes, Immobilized
  • Hepatitis A Vaccines
  • Peptides
  • Hyaluronic Acid
  • Chitosan
  • Trypsin