Abstract:
The
evaluation, analysis and development of an
oxygen-insensitive amperometric glucose biosensor and its
application in microbial batch culture are described. The
biosensor consisted of a graphite foil electrode modified with
glucose oxidase and 1,1'-dimethylferrocene, and operated via
mediated electron transfer from the enzyme to the electrode.
Initial evaluations illustrated several
operating
characteristics which would be
expected to cause problems in
continuous
monitoring applications, most notably sensor
instability and a progressive increase in response time. The
main
underlying causes of these unfavorable characteristics were
identified as enzyme loss, mediator loss and substrate diffusion
limitation within the electrode. As a consequence of these
insights, further development of the sensor was undertaken. A
number of different electrode materials and enzyme immobilization
techniques were tested, resulting in the development of a novel
immobilization procedure using a hexadecylamine coating to bind
'the activated carbohydrate residues of periodate-oxidized glucose
oxidase.
This improved the sensor lifetime and response time
under continuous operation.
Strategies for the reliable application of the
biosensor in fermentation
monitoring were evaluated. In-line
flow cell and
in_§itu membrane probe approaches were considered,
and the latter
approach was
preferred: Considerable attention
was devoted to
optimising the design of such probes. The best
design accommodated a three electrode configuration with a
multiple biosensor array. It was found necessary to allow for
periodic on-line calibration within the aseptically operating
probe. This configuration was successfully applied on-line to
monitor glucose in batch cultures of Escherichia coli.
