INTRODUCTIONProtein-immobilized substrates have been widely used in several applications for biosensing and bioseparation. Many kinds of sensors have been based on changes in colorimetric, fluorometric or luminometric signals derived from the immunoreactions of protein-immobilized sensor chips and particles. 1-4 Protein-immobilized substrates, such as column packings and magnetic beads, have also been used as selective materials that can rapidly and simply separate target biomolecules in solution.In the development of high-performance materials for biosensing and bioseparation, an effective blocking treatment is important to decrease nonspecific adsorption onto the surfaces of protein-immobilized substrates and to increase the dispersion stability of substrate particles. Poly(ethylene glycol) (PEG) is known as an excellent blocking agent. Because of the nonionic properties, hydrophilicity and large steric-exclusion effect of PEG, 5 PEGylated surfaces and nano-and microscale particles show excellent non-fouling properties with various molecules 6 and high-dispersion stabilities, 7-9 respectively. Furthermore, in our recent studies, we discovered that PEGylation improves the functioning of immobilized proteins on solid surfaces and particles. For example, the antigen-binding efficiencies of an anti-C-reactive protein antibody-immobilized gold sensor surface 10 and anti-ferritin antibody-immobilized latex particles 11 were improved by the co-immobilization of densely packed PEG layers, consisting of sulfhydryl-terminated PEGs and oligoamine-terminated PEGs. The substrate reactivity of glucose dehydrogenase-immobilized gold nanoparticles was improved by the co-immobilization of PEG/polyamine block copolymer, 8 and lipase/PEG-polyamine/glucose dehydrogenaseimmobilized gold nanoparticle hybrids showed almost the same initial enzymatic activity after five repeated thermal treatments at 58 1C for 10 min. 12 These results indicate that the co-immobilization of PEG