• Introduction

• XPS Characterization

• Proof of Crosslinking

• Biocompatibility of Prepared Membrane

• Separation of Papain Inhibitor from Potato Tuber

• Comparison of the Crosslinking Methods

• Separation of Fibronectin from Human Plasma

• Introduction
  
  Affinity membranes containing trypsin and papain immobilized as affinity ligands were prepared from glass fiber filters. The filters were first treated with a concentrated solution of H2SO4 + H2O2 (piranha solution), and then modified by silanization to introduce epoxyl or amino functional groups, which were further modified to carboxyl or aniline moieties, respectively. Onto the modified membranes, bovine serum albumin, trypsin and papain were immobilized as ligands. Three immobilization methods based on glutaraldehyde, carbodiimide and diazotization were used and compared to conclude that the one based on glutaraldehyde, which can form a crosslinking structure among the membrane, was the most efficient.

• XPS Characterization of Glass Surface Modification
  
  Besides the introduction of suitable functional groups, the silanization also helped to generate a uniform layer over the glass surface by capping the high potential sites, which stimulated irreversible protein adsorption when the membranes were used for filtration [31]. Figures 1 (b) and (c), show that after silanization, no metallic ions are present on the surface of the glass, which means that the ions present in Figure 1 (a) were all capped, and that the surface of glass was covered by a silane compound.
  

XPS Spectra of the glass fiber membranes

• Membrane Preparation
  
  The glass filter was first treated with piranha solution, and then modified with gamma-glycidoxypropyltriethoxysilaneg-glycidoxypropyltriethoxysilane to introduce amino groups.
  

Crosslinking of Glass Fibers with Glutaraldehyde

• Proof of Crosslinking
  
  The activities of the immobilized trypsin, prepared by the three methods (glutaraldehyde, diazotization and carbodiimide), are compared in the following figure (dynamic stability). One can conclude that the activity of the immobilized trypsin prepared via the carbodiimide method is comparable to that prepared via diazotization and that both decrease with the number of determinations (washings). These occurred because, in contrast to the glutaraldehyde method, no crosslinking which stabilizes the structure took place during immobilization.
  

• Biocompatibility of Prepared Membrane
  
  The stabilities of the affinity membranes are controlled by two important factors: (i) the resistance to the washing by the buffer (the dynamic stability), and (ii) the durability to denaturation (biocompatibility). The biocompatibility of the glass membrane (static stability) was investigated by determining the rate of denaturation of the immobilized trypsin. The figure shows that all three kinds of immobilized trypsin were very stable at room temperature, indicating a high biocompatibility of the glass membrane.
  

• Separation of Papain Inhibitor from Potato Tuber
  

• Comparison of the Crosslinking Methods
  
  Besides the glutaraldehyde method, the glass membranes were also crosslinked with the imine or ethylene bifunctional silane, and the effects were examined by the EMS
  

• Separation of Fibronectin from Human Plasma using Gelatin followed by Heparin Affinity Membranes