Surface immobilized enzymes for biosensing
Surface immobilized enzymes are widely used in biosensing applications. When enzyme molecules are deposited onto a surface, some molecules may adopt wrong orientations and subsequently lose their activity. We aim to develop a systematic approach to determine enzyme orientation after surface immobilization in order to understand the enzyme structure-function relationship. We have elucidated the effects of enzyme surface immobilization site location, number of immobilization sites, enzyme surface coverage, and substrate surface property on the orientation of surface immobilized enzymes. Sum frequency generation (SFG) vibrational spectroscopy has been extensively applied in this research, supplemented by attenuated total reflectance – FTIR (ATR-FTIR) spectroscopy, circular dichroism spectroscopy, and molecular dynamics simulations.