| Summary: | The
recently proposed bioassay procedure that is based on the substrate induced
quenching (SIQ) of an indicator fluorescence for the measurement of analyte
concentrations is evaluated. In this type of assay a enzynatic reaction and a
fluorescence
quenching interaction are coupled together. Typically, an appropriate
dehydrogenase enzyme reduces or oxidises the nicotinamide adenine dinucleotide
cofactor. The
change in the concentration of NADH results in variations in the excited
fluorophore population as observed through fluorescence intensity. This latter aspect is
used to monitor substrate (analyte) concentrations.
Results on the
investigation of the substrate induced quenching bioassay method and
possibilities of using it as the basis of (i) a novel enzyme bioassay technique and (ii) a
novel
bioprobe format are presented. Ethanol was chosen as the model analyte, and a
new
assay procedure for its measurement was developed.
A
generic theoretical relation is discussed for the observed assay kinetics of substrate
induced
quenching (SIQ) and a model is described that includes the effects due to
dynamic/static quenching of the fluorophore by either the enzyme substrate or product.
The
validity of the derived model is shown by comparison with experimental results for a
SIQ based ethanol assay. The option of running the dehydrogenase reaction so as to
consume NADH rather than
generate it is also investigated. In order to demonstrate this
approach acetaldehyde was chosen as the model analyte, and a assay procedure for its
measurement was
developed.
The
potential of the SIQ technique for incorporation into biosensor based upon a
'reservoir' format was demonstrated through the development of custom optical
instrumentation and resevoir flowcell. Applicability of the SIQ technique to other
biosensor formats such as flow-injection analysis and 'dry reagent' technology is
discussed.
The overall
applicability of the SIQ technique is assessed through the generation of a
number of SIQ assays on the following substrates: ethanol, glucose, glucose-6-
phosphate, L-glutamic acid, isocitric acid, acetaldehyde, pyruvic acid, ot-ketoglutaric
acid, and oxalacetic acid.
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