Title:
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The amperometric study of enzymes and their substances
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This thesis describes an investigation into the use of
amperometric techniques for the detection of enzyme catalysed
reactions.
Enzymes catalyse a vast number of biological reactions
and the detection of their activity can lead to the
development of analyses for their substrates.The specificity
with which the enzyme recognises its substrate is unique and
cannot be easily mimicked by chemical catalysis.Enzymatic
analyses can therefore be highly discriminating, they are
also often the best methods for biological analyses as they
can be carried out under mild conditions.
The isolation of enzymes for use in vitro can also
enable substrate analogues, which the molecule would not
normally encounter, to be detected using that enzyme.
Traditional methods of detecting enzyme activity rely on
expensive equipment and skilled operation.Current trends in
analysis, especially in the medical field, are towards
methods which can be used away from laboratories and devices
capable of such analyses are called biosensors.Work carried
out in this thesis uses the electrochemical technique of
amperometry for the detection of enzyme catalysed reactions
The simple instrumentation and rapidity associated with this
technique facilitates the development of biosensors from the
analyses described.
The thesis examines three different systems.The first is
a specific sensor for paracetamol (acetaminophen), the second
a more widely applicable assay for dehydrogenases .This
analysis is based on the amperometric detection of NADH using
the mediated electrochemistry of the enzyme diaphorase.The
same system has also been used to study the kinetics of the
overall reaction which are compared with a kinetic analysis
carried out by conventional methods.
The final assay is for the detection of catalase, this
extremely active enzyme is also conjugated to antibody
molecules such that an enzyme linked immunosorbent assay
(ELISA) with amperometric detection can be developed.
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