Chemical and biological sensors based on electroactive inorganic polycrystals - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 427

cupric hexacyanoferrate is useful for carbon paste biosensors [162]. An apparently fi rst
publication on Prussian blue-based immuno-sensors appeared recently [173].
13.5.3 Immobilization of the enzymes using non-conventional media
A necessary part of biosensor construction is the biorecognition element immobilized
on the top surface of the transducer. Thus, immobilization of biocomponents (enzymes
in the case of the present review) is an important and crucial point of biosensor con-
struction. Certainly, enzyme immobilization is a widely investigated area, which can
even be recognized as a precursor to modern biotechnology. This undoubtedly makes it
impossible to review in a single chapter. Hence, only the original protocol for enzyme
immobilization providing substantial improvement of analytical performances of the
resulting biosensors will be presented.
Among various enzyme immobilization protocols, entrapment in polymer mem-
branes is a general one for a variety of transducers. Formation of a membrane from
a solution of already synthesized polymer is simpler and reproducible compared to
chemical polymerization. The simplicity of this immobilization procedure should pro-
vide reproducibility for the resulting biosensors; the latter is strongly required for mass
production.
Since the polymer has to be water-insoluble, its complete solution may occur only
in an organic solvent. Casting the polymer from such a solution obviously can improve
the properties of the resulting membrane. Thus, to prepare an enzyme containing cast-
ing solution the protein has to be exposed to organic solvent.
The investigation of enzymes in water-miscible organic solvents trivially called
“non-aqueous enzymology” about 20 years ago became an independent part of modern
biochemistry and enzymology [174-176]. In concentrated organic solvents, with the
water content less than 10-15%, the enzymes are rather stable and can even retain their
activity [176, 177]. Recent studies even demonstrated improvement of the enzyme
activity in concentrated organic solvents [178].
The methodology of the proposed immobilization approach [164, 179] is, however,
quite different from “non-aqueous enzymology”. Though during an immobilization
procedure, enzymes have to be exposed to organic solvents, their activity is required
only in aqueous solution in which resulting biosensors are operated. Hence, it is only
important that the enzymes are able to retain their catalytic properties after exposure to
organic solvents.
13.5.3.1 Tolerance of the enzymes to organic solvents
It was thus necessary to investigate what happens with the enzyme activity after expo-
sure to an organic solvent. Figure 13.7 illustrates the remaining activity of alcohol
dehydrogenase and glucose oxidase after 30 minutes in concentrated ethanol, isopro-
panol, and acetonitrile, related to initial enzyme activity in aqueous solution. As the
water content in the water-organic mixture is increased, the activity of the enzymes is
also initially increased, but further additions of water result in its decrease. The general