Probing the Distribution of Immobilized Enzyme Within Hierarchical Structures

3 PROBING THE DISTRIBUTION OF IMMOBILIZED ENZYME WITHIN HIERARCHICAL STRUCTURES 

Fluorescence microscopy can be used to track the distribution of enzyme within an immobilization matrix, particularly if the electrode architecture is a three-dimensional macroporous scaffold. Specifically, fluorophores can be tethered to a protein (e.g., Alexa Fluor 546) or included as fluorescent dyes (e.g., Nile red), added to the immobilization material, and excited at the appropriate wavelength for visualization after the immobilization procedure. Malatc dehydrogenase (MDH), for example, was encapsulated in 2009 by Martin et al. in chitosan polymer, either in its native state or following hydrophobic modification by formalion of an amide with butyric acid (butyl-modified) or a-linoleic acid (ALA) [27]. 

Fluorescence can also provide unique information on the chemical microenvironment surrounding the encapsulated enzyme. For example, Martin et al. 

FIGURE 12.3 Distribution ratio of the emission intensity below 620 nm to the total emission intensity (each data point represents a single pixel ina512x512 confocal image). (Reproduced with permission from Ref. [27]. Copyright 2009, American Chemical Society.) 

---