The immobilization of enzymes on solid supports is widely used in many applications including biosensors, antifouling coatings, food packaging materials and biofuel cells. Enzymes tend to lose their activity when in contact with a support surface, a phenomenon that has been attributed to unfavorable orientation and (partial) unfolding. In this work, specific immobilization of 6-phospho-β-galactosidase (β-Gal) through a unique cysteinyl residue was achieved on self-assembled monolayer (SAM) containing maleimide end groups and oligo ethyleneglycol spacer segments. A systematic means to characterize interfacial orientation of immobilized enzymes has been developed using combined studies with sum frequenc... More
The immobilization of enzymes on solid supports is widely used in many applications including biosensors, antifouling coatings, food packaging materials and biofuel cells. Enzymes tend to lose their activity when in contact with a support surface, a phenomenon that has been attributed to unfavorable orientation and (partial) unfolding. In this work, specific immobilization of 6-phospho-β-galactosidase (β-Gal) through a unique cysteinyl residue was achieved on self-assembled monolayer (SAM) containing maleimide end groups and oligo ethyleneglycol spacer segments. A systematic means to characterize interfacial orientation of immobilized enzymes has been developed using combined studies with sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance (ATR) - FTIR spectroscopy. The possible orientations of the immobilized β-Gal were determined, which are well correlated to the tested activity of β-Gal. This study will impact the development of an increasingly wide range of devices that use surface-immobilized enzymes as integral component with improved functions, better sensitivity, enhanced stability, and longer shelf-life.
