Enzyme distribution and secondary structure of sol-gel immobilized glucose oxidase by micro-attenuated total reflection FT-IR spectroscopy.

Glucose oxidase (GOD) immobilized into sol-gel matrices was studied by using Micro-Attenuated Total Reflection Fourier Transform Infrared (micro-ATR FT-IR) spectroscopy in order to characterize enzyme distribution and secondary structure in systems with valuable potentialities in amperometric and optical biosensing. Spectra were acquired in the 4000-600 cm(-1) frequency region and the analysis of specific fingerprints in the FT-IR spectra evidenced that the enzyme was actually immobilized in the matrix. The enzyme spatial distribution was obtained by examining the amide I and amide II band region of spectra from defined sample positions. The deconvolution of the amide I band in terms of lorentzian functions provided information on the secondary structure of the immobilized GOD. By this approach a macroscopic preservation of GOD activity upon immobilization was evidenced along with the existence of some matrix sites with locally inactivated GOD. To our knowledge this is the first example of point-by-point characterization of conformational changes of immobilized enzyme by means of micro-ATR infrared spectroscopy, thus confirming that this technique can be usefully employed for a non- or minimally-invasive detailed micro-characterization of catalytic supports in order to improve their functionality.