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Philipps-Universität Marburg
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Abstract
The aim of this work was the development of an amperometric biosensor
for cyanide by the use of a three-step reaction cascade involving two
enzymes. In the first step of this cascade, the enzyme cyanidase
(EC 3.5.5.1) hydrolyzes cyanide to formic acid and ammonia. In the
following, formate, the corresponding base of formic acid, is oxidized
to carbon dioxide by catalysis of formate dehydrogenase (EC 1.2.1.2),
while NAD, serving as cosubstrate, is reduced to NADH. In the third
step, the oxidized form of a redox mediator reacts with NADH, yielding
NAD and the reduced form of the mediator. Amperometric detection is
possible for formate on the first, NADH on the second and the reduced
form of the mediator on the third step of the cascade.
Technically, two different sensor setups were used. On the one hand, a
flow-through system was employed. It consisted of an injection valve,
enzyme cartridges and a flow cell with amperometric electrode chips.
On the other hand, gas phase experiments were carried out by placing a
mixture onto an electrode chip. The mixture contained the enzymes and
reagents necessary for the reaction cascade. The electrode chip was
put in the gas phase of a sealed vessel. An acid component resided at
the bottom of this vessel. Addition of cyanide containing samples into
the acid resulted in protonation of cyanide, leading to hydrogen
cyanide. Hydrogen cyanide then entered the gas phase and reached the
mixture covering the electrode chip.
Detection of formate at the first step of the cascade was possible
solely by platinum working electrodes. In a flow-through setup,
cyanidase immobilized in a cartridge was not able to completely
hydrolyze the cyanide in samples. The remaining cyanide interacted
with the platinum electrode, interfering with measurements. A removal of
non-hydrolyzed cyanide by formation of metal complexes was not
successful.
Detection of NADH was feasible by the use of a flow-through setup. The
limit of detection and the limit of quantification were determined to
be 0.7 and 2.4 µM, respectively. The linear range extended from the
limit of detection up to 1 mM. Nitriles and sulfide did not interfere,
while thiocyanate showed a moderate interference. However, formate is
expected to cause disproportionate signals by design. Estimation of
cyanide in real samples with complex matrices such as plant extracts
were possible. The correctness of the determination of cyanide
concentrations in extracts of leaves of cherry laurel were checked by
the use of the DIN method 38405-13 as a reference. With an average
deviation of -4,8%, the sensor results were in good conformance with
the reference results. Moreover, a good storage stability was found.
After more than three months, no decrease of sensitivity could be
observed.
In contrast, gas phase measurements proved to be problematic.
Additions of cyanide-free liquids into the acid component induced
signals similar to those of cyanide containing samples, supposedly
caused by the influence of changes in water vapor pressure. Meldola
Blue as a redox mediator together with graphite working electrodes
showed to be the only combination able to differentiate between
cyanide free and cyanide containing samples (millimolar concentration
range). During these experiments, concentrations of hydrogen cyanide
in gaseous phase were between 2 and 800 ppm. Other mediators such as
ferricyanide or quinones were found to be unsuitable. Signal heights
were hardly reproducible, even when using Meldola Blue. Furthermore,
the rate of signal response changed in the course of experiments.
Also, the orientation of the electrode chip influenced the signal
trend. The mixture could not be fixated on the chip. Chemical and
electrochemical modifications of the working electrode were
dissatisfying. To successfully develop a practicable amperometric gas
sensor, further investigations are necessary. Yet, the three-step
reaction cascade basically seems to be suited for measurements in
gaseous phase.
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Dates
Created: 2010Issued: 2010-07-08Updated: 2011-08-10
Faculty
Fachbereich Pharmazie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
AmperometryCyanidHydrogen cyanideCyanidaseBiosensorCyanideCyanidase
DFG-subjects
BiosensorAmperometrieBlausäure
DDC-Numbers
540
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Ketterer, Lothar Rudolf (141524634): Entwicklung eines amperometrischen Biosensors für Cyanid. : Philipps-Universität Marburg 2010-07-08. DOI: https://doi.org/10.17192/z2010.0167.
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This item has been published with the following license: In Copyright