Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination
Aim. The development and optimization of the amperometric biosensor for pyruvate determina-tion. Methods. Immobilized pyruvate oxidase was used as a biorecognition element of the biosen-sor, a platinum disc electrode- as an electrochemical transducer. Results. Different variants of immobilization of...
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Інститут молекулярної біології і генетики НАН України
2018
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irk-123456789-1542612019-07-07T12:38:49Z Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination Knyzhnykova, D.V. Topolnikova, Ya.V. Kucherenko, I.S. Soldatkin, O.O. Molecular and Cell Biotechnologies Aim. The development and optimization of the amperometric biosensor for pyruvate determina-tion. Methods. Immobilized pyruvate oxidase was used as a biorecognition element of the biosen-sor, a platinum disc electrode- as an electrochemical transducer. Results. Different variants of immobilization of pyruvate oxidase were tested and the optimal one was chosen for the creation of a biorecognition element of the biosensor. Optimal concentrations of cofactors for the best per-formance of the pyruvate oxidase-based biosensor were selected. The developed biosensor dem-onstrated a high sensitivity to pyruvate and wide linear range of work. High selectivity of the pro-posed biosensor towards electrically active substances and other substrates present in real samples was shown. The biosensor is characterized by high signal reproducibility and operational stability over two weeks. Conclusions. The highly selective amperometric biosensor for determination of pyruvate in biological samples has been developed. Its analytical characteristics are studied. The biosensor can be further used for the pyruvate analysis in blood serum. Мета. Розробка та оптимізація роботи амперометричного біосенсора для визначення пірувату. Методи. Застосовано амперометричний біосенсор з іммобілізованою піруватоксидазою як біоселективний елемент та платинові дискові електроди як перетворювачі. Результати. Перевірено різні варіанти іммобілізації піруватоксидази та обрано оптимальний для створення біоселективного елементу біосенсора. Підібрано оптимальні концентрації кофакторів для найкращої роботи біосенсора на основі піруватоксидази. Розроблений біосенсор демонстрував високу чутливість до пірувату та широкий лінійний діапазон роботи. Було показано високу селективність запропонованого біосенсора відносно електроактивних речовин та інших субстратів, що можуть бути присутніми в реальних зразках. Біосенсор характеризується високою відтворюваністю сигналу та операційною стабільністю протягом 2 тижнів. Висновки. Розроблено високоселективний амперометричний біосенсор для визначення пірувату у біологічних зразках та досліджено його аналітичні характеристики. В подальшому даний біосенсор може бути використано для визначення пірувату у сироватці крові. Цель. Разработка и оптимизация работы амперометрического біосенсора для определения пирувата. Методы. Использовано амперометрический біосенсор с иммобилизованной пируватоксидазой как биоселективным элементом и платиновые дисковые электроды как преобразователи. Результаты. Проверено разные варианты иммобилизации пируватоксидазы и выбрано оптимальный для создания биоселективного элемента биосенсора. Подобрано оптимальные концентрации кофакторов для наилучшей работы биосенсора на основе пируватоксидазы. Разработанный биосенсор демонстрировал высокую чувствительность к пирувату и широкий линейный диапазон работы. Было показано хорошую селективность предложенного биосенсора относительно электроактивных веществ и других субстратов, которые могут присутствовать в реальных образцах. Биосенсор характеризуется высокой воспроизводимостью сигнала и операционной стабильностью на протяжении двух недель. Выводы. Разработан высокоселективный амперометрический биосенсор для определения пирувата в биологических образцах и исследованы его аналитические характеристики. В дальнейшем данный биосенсор может быть использован для определения пирувата в сыворотке крови. 2018 Article Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination / D.V. Knyzhnykova, Ya.V. Topolnikova, I.S. Kucherenko, O.O. Soldatkin // Вiopolymers and Cell. — 2018. — Т. 34, № 1. — С. 14-23. — Бібліогр.: 9 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00096C http://dspace.nbuv.gov.ua/handle/123456789/154261 543.06 + 577.15 + 543.553 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| language |
English |
| topic |
Molecular and Cell Biotechnologies Molecular and Cell Biotechnologies |
| spellingShingle |
Molecular and Cell Biotechnologies Molecular and Cell Biotechnologies Knyzhnykova, D.V. Topolnikova, Ya.V. Kucherenko, I.S. Soldatkin, O.O. Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination Вiopolymers and Cell |
| description |
Aim. The development and optimization of the amperometric biosensor for pyruvate determina-tion. Methods. Immobilized pyruvate oxidase was used as a biorecognition element of the biosen-sor, a platinum disc electrode- as an electrochemical transducer. Results. Different variants of immobilization of pyruvate oxidase were tested and the optimal one was chosen for the creation of a biorecognition element of the biosensor. Optimal concentrations of cofactors for the best per-formance of the pyruvate oxidase-based biosensor were selected. The developed biosensor dem-onstrated a high sensitivity to pyruvate and wide linear range of work. High selectivity of the pro-posed biosensor towards electrically active substances and other substrates present in real samples was shown. The biosensor is characterized by high signal reproducibility and operational stability over two weeks. Conclusions. The highly selective amperometric biosensor for determination of pyruvate in biological samples has been developed. Its analytical characteristics are studied. The biosensor can be further used for the pyruvate analysis in blood serum. |
| format |
Article |
| author |
Knyzhnykova, D.V. Topolnikova, Ya.V. Kucherenko, I.S. Soldatkin, O.O. |
| author_facet |
Knyzhnykova, D.V. Topolnikova, Ya.V. Kucherenko, I.S. Soldatkin, O.O. |
| author_sort |
Knyzhnykova, D.V. |
| title |
Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination |
| title_short |
Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination |
| title_full |
Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination |
| title_fullStr |
Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination |
| title_full_unstemmed |
Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination |
| title_sort |
development of pyruvate oxidase-based amperometric biosensor for pyruvate determination |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2018 |
| topic_facet |
Molecular and Cell Biotechnologies |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/154261 |
| citation_txt |
Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination / D.V. Knyzhnykova, Ya.V. Topolnikova, I.S. Kucherenko, O.O. Soldatkin // Вiopolymers and Cell. — 2018. — Т. 34, № 1. — С. 14-23. — Бібліогр.: 9 назв. — англ. |
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14
D. V. Knyzhnykova, Ya. V. Topolnikova, I. S. Kucherenko
© 2018 D. V. Knyzhnykova et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf
of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any
medium, provided the original work is properly cited
UDC 543.06 + 577.15 + 543.553
Development of pyruvate oxidase-based amperometric biosensor
for pyruvate determination
D. V. Knyzhnykova1,2, Ya. V. Topolnikova1, I. S. Kucherenko1, O. O. Soldatkin1,2
1 Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
2 Institute of High Technologies, Taras Shevchenko National University of Kyiv
2, korp.5, Pr. Akademika Hlushkova, Kyiv, Ukraine, 03022
kucherenko.i.s@gmail.com
Aim. Development and optimization of the amperometric biosensor for pyruvate determina-
tion. Methods. Immobilized pyruvate oxidase was used as a biorecognition element of the
biosensor, a platinum disc electrode, as an electrochemical transducer. Results. Different
variants of immobilization of pyruvate oxidase were tested and the optimal one was chosen
for the creation of a biorecognition element of the biosensor. Optimal concentrations of cofac-
tors for the best performance of the pyruvate oxidase-based biosensor were selected. The
developed biosensor demonstrated a high sensitivity to pyruvate and wide linear range. High
selectivity of the proposed biosensor towards electrically active substances and other substrates
present in real samples was shown. The biosensor is characterized by high signal reproducibil-
ity and operational stability over two weeks. Conclusions. The highly selective amperometric
biosensor for determination of pyruvate in biological samples has been developed. Its ana-
lytical characteristics were studied. The biosensor can be further used for the pyruvate analy-
sis in blood serum.
K e y w o r d s: pyruvate, pyruvate oxidase, amperometric biosensor.
Introduction
Pyruvate is a simple keto acid, which plays a
central role in the metabolism and carbohy-
drate conversions. Pyruvate is a key metabolite
in a number of biochemical transformations
associated with the catabolism in mitochon-
dria. The main metabolic pathways of pyruvate
are aerobic oxidation to oxaloacetate or an-
aerobic conversion to lactate. Pyruvate oxida-
tion involves the enzyme pyruvate dehydroge-
nase and is an intermediate step between gly-
colysis and Krebs cycle.
The pyruvate concentration in the blood
normally ranges from 40–50 to 100 μmol/L
[1, 2]. The pyruvate level increases when the
aero bic processes intensify and at insufficient
pyruvate utilization in the pyruvate dehydro-
Molecular and Cell
Biotechnologies
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2018. Vol. 34. N 1. P 14–23
doi: http://dx.doi.org/10.7124/bc.00096C
mailto:kucherenko.i.s@gmail.com
15
Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination
genase complex. A higher pyruvate concentra-
tion is observed in the cases of vitamin B1
deficiency, respiratory alkalosis (a sharp de-
crease in the level of carbon dioxide in the
blood, accompanied by an increase in pH),
arsenic and mercury poisoning, and liver pa-
thologies such as alcoholic cirrhosis, hepati-
tis, etc. [3].
An increased by 2-2.8 times concentration
of pyruvate is also found in the serum and
saliva of oral cancer patients. The evaluation
of pyruvate concentration is considered as a
new method of cancer screening [2, 4].
Additionally, an increased content of pyruvate
in tissues typically indicates an imbalance in
the systems of oxygen supply and consump-
tion. The pyruvate concentration in the blood
is an informative laboratory evidence of the
adequacy of blood supply and tissue oxygen-
ation or hypoxia.
In the clinical practice of emergency ther-
apy, the pyruvate monitoring has not been
implemented yet due to the selectivity prob-
lems – the pyruvate concentration in the blood
is low whereas the concentration of electroac-
tive interferents is quite high. The development
of electrochemical biosensors can solve this
problem.
To date, there are a number of biosensors
for pyruvate measuring intended for clinical
use. Thus, Gajovic et al. developed the biosen-
sor based on recombinant pyruvate oxidase for
the pyruvate determination in serum [3]. For
the protection against intense interference from
ascorbate, the electrode surface was coated
with a dialysis membrane based on acetate and
nitrocellulose. The biosensor demonstrated a
limit of pyruvate detection of 30 μM in calf
serum, which, according to the authors, is suf-
ficient for continuous monitoring of pyruvate
in organs and tissues at hypoxia. Arai et al.
immobilized pyruvate oxidase in the layer of
a conductive redox polymer poly(mercapto-p-
benzoquinone) by electropolymerization on
the electrode surface [5]. Here, the polymer
molecules functioned as a chain for the elec-
trons transfer between the enzyme active cen-
ter and the electrode surface. The dynamic
range of this biosensor was from 1 μM to
2 mM. Akyilmaz et al. developed a pyruvate
oxidase-based biosensor to measure pyruvate
and phosphate for clinical use. The enzyme
was immobilized in gelatin and fixed by cross-
linking with glutaraldehyde [6].
Unfortunately, none of the known biosen-
sors for the pyruvate determination was ac-
complished and commercialized. Therefore,
the purpose of this work was to develop an
amperometric biosensor for quantitative anal-
ysis of pyruvate concentration in biological
fluids. This is the extension and continuation
of our previous work, where we described the
amperometric biosensor system for the deter-
mination of lactate and pyruvate [7].
Materials and Methods
Materials
In this work we used pyruvate oxidase
(PyrOx) from Aerococcus sp. (EC 1.2.3.3),
activity 54 U∙mg-1, sodium pyruvate, bovine
serum albumin, glycerol, polyvinyl alcohol
photopolymer containing styrylpyridine
groups (PVA-SbQ), magnesium nitrate, 25%
aqueous glutaraldehyde (GA) solution, ace-
tylcholine chloride, choline chloride, gluta-
mate chloride and HEPES from Sigma-
Aldrich (USA), thiamine pyrophosphate
16
D. V. Knyzhnykova, Ya. V. Topolnikova, I. S. Kucherenko et al.
(TPP) (lyophilisate for injection solutions)
from Biofarma (Ukraine), and potassium di-
hydrogen phosphate (KH2PO4) were from
Helicon (Russia). Other inorganic compounds
used in the work were of domestic production
and had reagent purity grade.
Microparticles of silicalite were synthesized
artificially in accordance with the method de-
scribed in the previous paper [8].
Design of amperometric transducers
Platinum disk electrodes were manufactured
in our laboratory using the following techno-
logy. 3 mm long platinum wire, 0.5 mm in
diameter, was soldered with low-temperature
Wood’s alloy to the silver wire and placed into
a glass capillary with outer diameter 3.5 mm.
The end of capillary with inserted platinum
wire was sealed by soldering and used as a
sensitive surface of the transducer. The elec-
trode working part with inserted platinum wire
was periodically treated with sandpaper and
aluminum microparticles to restore the sensi-
tivity of transducer.
Preparation of bioselective elements
A bioselective element of the biosensor was
obtained by immobilization of the enzymes
and auxiliary substances onto the surface of
amperometric transducer. The initial solution
consisted of 20% of PyrOx, 5% of BSA, 10%
of glycerol in 100 mM phosphate buffer,
pH 6.5. Glycerol was added to stabilize the
enzymes during their immobilization as well
as to prevent early drying of the membrane
and improve its adhesion to the transducer
surface. Immobilization of PyrOx was per-
formed in three different ways: cross-linking
with glutaraldehyde, adsorption on silicalite,
and entrapment into polymer PVA-SbQ.
Measurement procedure
A three-electrode scheme of the amperometric
analysis was used. The working amperometric
transducers, an auxiliary platinum electrode,
and an Ag/AgCl reference electrode were con-
nected to the potentiostat PalmSens
(PalmInstruments BV, the Netherlands). The
8-channel device CH-8 multiplexer (Palm Inst-
ru ments BV, the Netherlands) connected to the
potentiostat allowed receiving the signals si-
multaneously from eight working electrodes,
but usually only two-three working electrodes
were in operation simultaneously. The distance
between the auxiliary platinum electrode and
all working electrodes was stable over the
entire measurement procedure and was ap-
proximately 5 mm.
Chronoamperometric measurements (“am-
perometric detection” technique) were carried
out at room temperature in an open measuring
cell of 2.5 ml volume at permanent stirring and
constant potential of +0.6 V vs Ag/AgCl refe-
rence electrode. The 50 mM phosphate buffer
(KH2PO4-Na2HPO4), pH 6.5, was used as a
working buffer, unless different stated. TPP
and magnesium ions were necessary for the
PyrOx activity and were added to the working
buffer (several concentrations were used, as
stated in the text). The biosensor connected to
the potentiostat was immersed into the cell
with working buffer and kept for several min-
utes with applied potential until stable baseline
was obtained. Then certain aliquots of the
model pyruvate solution were added and the
biosensor’s responses were recorded using the
17
Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination
computer program. All experiments were car-
ried out in at least three replicates.
Results and Discussion
The operation of amperometric biosensor for
pyruvate determination is based on the enzy-
matic reaction (1) occurring in the bioselective
membrane:
The reaction results in pyruvate oxidation
with the formation of electrochemically active
hydrogen peroxide. Application of a positive
potential on the electrode stimulates the reac-
tion of hydrogen peroxide decomposition (2)
and generation of electrons, which are di-
rectly registered using the amperometric trans-
ducer.
Н2О2
+ 0.6 V
2Н+ + О2 + 2е- (2)
Selection of the optimal working potential
Firstly, influence of the applied potential on
the current from the platinum disk electrodes
was investigated. The potential directly influ-
enced oxidation (or reduction) of hydrogen
peroxide on the electrode surface, and thus the
biosensor responses were greatly dependent
on the potential. We studied the biosensor
responses in the potential range from –0.5 V
to +0.9 V with 0.1 V step vs Ag/AgCl refer-
ence electrode (Fig. 1).
It was found that the responses to H2O2
could be recorded at any potential, but the
highest response values were obtained at
+0.6 V. Relatively high responses were ob-
served also at 0 V, but with negative current
due to hydrogen peroxide reduction. This re-
sult could be very promising since at 0 V [an]
impact of interfering substances is much lo wer,
but the noise of baseline was very high in this
case and accurate detection of target substanc-
es was impossible. Thus for the further re-
search 0.6 V was taken as the optimum value.
Optimization of the immobilization con-
ditions for PyrOx
An important step in the bioselective mem-
brane formation is immobilization of biologi-
cal material on the transducer surface with
maximum maintenance of the enzyme activity.
The following methods of immobilization were
tested: cross-linking using GA, adsorption on
a silicalite layer, entrapment in polymer PVA-
SbQ. The same amount of enzyme was used
to compare different methods.
pyruvate + phosphate + Н2О + О2
Pyruvate oxidase
acetyl phosphate + H2O2 + CO2 (1)
TPP, FAD, Mg2+
Fig. 1. Dependence of the amperometric transducer re-
sponses on applied potential. Hydrogen peroxide concen-
tration 1 mM. Measurements in 50 mM phosphate buffer,
pH 6.5
18
D. V. Knyzhnykova, Ya. V. Topolnikova, I. S. Kucherenko et al.
PyrOx immobilization with GA was inves-
tigated firstly. The enzyme solution was mixed
with low-concentration glutaraldehyde aque-
ous solution in [the] 1:1 ratio and immedi-
ately afterwards the obtained mixture was de-
posited onto the transducer’s surface. The bi-
oselective elements with different GA mass
fraction (0.15%, 0.35% and 0.25%) were used.
Then the transducers were kept in the open air
at room temperature for 15 min to allow cross-
linking of PyrOx and BSA, and washed with
the working buffer afterwards.
The second method of enzyme immobiliza-
tion was adsorption on silicalite. The silicalite
suspension (mass fraction 10%) in 50 mM
phosphate buffer, pH 6.5, was deposited onto
the transducer’s sensitive region. The latter
was heated to 100 °C for 5 min to improve the
attachment of silicalite to the surface. Next,
the transducers were cooled down and PyrOx
solution was deposited over the obtained sili-
calite layer. Afterwards, the transducer was
kept in the air at room temperature until com-
plete drying (15 min).
The third immobilization method was [the]
PyrOx entrapment in photopolymerized PVA-
SbQ. To form the membrane, PyrOx solution
and PVA-SbQ solution (a mass fraction 13.3%)
were mixed in the 1:1 ratio; the obtained mix-
ture was deposited onto the transducer surface
and exposed for 25 min to UV irradiation with
the ultraviolet lamp.
Analytical characteristics of [the] biosen-
sors based on different methods of immobiliza-
tion (sensitivity, linear range of pyruvate de-
termination, [a] lower limit of detection, [an]
upper limit of dynamic range, noise and drift
of the baseline) are shown in the Table 1. It
can be seen that the biosensors with PyrOx
adsorption on silicalite and PyrOx photopoly-
merization in PVA-SbQ had the widest linear
range of detection. The biosensors with im-
mobilization in a GA drop (mass fraction
0.15%) and photopolymerization in PVA-SbQ
demonstrated the highest sensitivity to pyru-
vate whereas the biosensors using PyrOx im-
mobilization via GA (mass fractions 0.25%
and 0.35%) were almost insensitive to pyru-
vate. The greatest noise of the baseline was
observed for the biosensors with the enzyme
immobilization via GA (0.15% GA), whereas
the lowest baseline noise – for those with
PyrOx adsorption on silicalite. An advantage
of the biosensors with PyrOx immobilization
Table 1. Comparison of analytical characteristics of biosensors created using different methods of
immobilization
Analytical characteristics
Type of immobilization
GA 0.15% GA 0.25% GA 0.35% Adsorption
on silicalite
Photopolymeri-zation
in PVA-SbQ
Sensitivity, nA/mM 22.1 1.5 2.9 11.5 23.7
Linear range, mM 0.15-5 0.9-3 0.3-11 0.1-7 0.01-5
Lower limit of detection, µM 17.7 308 170 6.0 5.1
Upper limit of dynamic range, mM 9.2 3.6 17.0 8.1 6.8
Noise of baseline, nA 0.18 0.14 0.13 0.06 0.09
Drift of baseline, nA/min 0.1 0.15 0.07 0.05 0
19
Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination
in a GA drop (0.35% GA) was the largest
value of the upper limit of dynamic range.
Of all methods of enzyme immobilization,
photopolymerization of PyrOx in PVA-SbQ
was characterized by the highest sensitivity to
pyruvate, the lowest limit of detection and the
best operational stability; therefore, in further
research we used this method.
Influence of cofactor concentrations
As known, the reaction of pyruvate oxidation
to acetyl phosphate and hydrogen peroxide
occurs in the presence of additional substan ces:
phosphate as the second substrate, TPP and
bivalent magnesium cation as cofactors of
PyrOx. Mg2+ plays essential role in the bin ding
of TPP, thus the Mg2+-TPP complex is formed,
which is required for PyrOx. Therefore, it was
necessary to study the impact of each sub-
stance and to determine their optimal concen-
trations.
First, the influence of TPP concentration on
the biosensor response to pyruvate was stu-
died. In the experiment, TPP in concentrations
from 1 μM to 2500 μM was added to the work-
ing buffer. As seen (Fig. 2), the response in-
creased up to 500 μM, so this concentration
was taken in further work as optimal.
To find the optimal concentration of Mg2+,
the biosensor sensitivity to pyruvate was test-
ed in the range 20 – 2400 μM Mg2+. As seen
from Fig. 3, the highest response was observed
at the magnesium concentration of 120 μM
whoch was taken as optimal in the further
experiments.
The value of biosensor response depends
also on the concentration of the phosphoric
acid ions, which are the substrate in the PyrOx-
catalyzed enzymatic reaction. There fo re, the
effect of the phosphate concentration in the
buffer solution on the biosensor response was
investigated. The developed biosensor for py-
Fig. 2. Dependence of biosensor response on TPP con-
centration. Concentration of pyruvate was 500 µM, mag-
nesium – 5 mM. Measurements were carried out in
50 mM phosphate buffer, pH 6.5, at a constant potential
of +0.6 V vs Ag/AgCl re fe ren ce electrode
Fig. 3. Dependence of biosensor response on magnesium
ions concentration. Concentration of pyruvate was
250 µM, TPP – 0.5 mM. Measurements were performed
in 50 mM phosphate buffer, pH 6.5, at a constant poten-
tial of +0.6 V vs Ag/AgCl reference electrode
20
D. V. Knyzhnykova, Ya. V. Topolnikova, I. S. Kucherenko et al.
ruvate analysis is planned to be combined with
other biosensors, in which HEPES-based buf-
fer is used as a working buffer. Therefore,
operation of the PyrOx-based biosensor was
investigated in the HEPES working buffer
containing ions of phosphoric acid of various
concentrations (1 – 100 mM). As seen (Fig. 4),
an increase in the phosphoric acid concentra-
tion up to 20 mM caused essential increase of
the biosensor response values whereas no
changes occurred at higher phosphoric acid
concentration. Therefore, the working buffer
with 20 mM phosphates was used in further
experiments
Analytical characteristics of the biosensor
The biosensor main analytical characteristics
were determined under optimized working
conditions. A typical calibration curve is shown
in Fig. 5. As known, the lower limit of detec-
tion corresponds to the substrate concentration,
at which the biosensor response is three-times
higher than the value of baseline noise. The
limit of detection of pyruvate was determined
as 8.1 μM and slightly differed for each indi-
vidual biosensor. The linear range of pyruvate
determination was 0.01 – 5 mM], sensitivity
to pyruvate was 31.06 nA/mM. The linear part
of calibration curve is described by the equa-
tion І = 31.06*С + 1.88 (R2 = 0,989), where I
is the current value corresponding to the
steady-state res ponse (nA), C – pyruvate
concentra ti on (mM).
Reproducibility of responses is one of the
main working characteristics of biosensors.
However, the washing-out of immobilized
components from the bioselective membrane
and the enzyme inactivation are observed in
the course of operation. Therefore, the next
step in our work was to test the reproducibil-
ity of the biosensor response during several
hours of continuous work. The biosensor re-
sponses to 500 µM pyruvate were measured
Fig. 4. Dependence of the biosensor response on concen-
tration of phosphoric acid ions. Concentration of pyru-
vate was 500 µM, TPP – 500 μM, Mg2+ – 120 μM. Mea-
surements were carried out in 25 mM HEPES buffer,
pH 7.4, at a constant potential of +0.6 V vs Ag/AgCl re-
fe rence electrode
Fig. 5. Dependence of response of PyrOx-based biosen-
sor on pyruvate concentration. Concentration of mag ne-
sium ions – 120 μM, TPP – 0.5 mM. Measurements
were carried out in 50 mM phosphate buffer, pH 6.5, at
a constant potential of +0.6 V vs Ag/AgCl reference
electrode
21
Development of pyruvate oxidase-based amperometric biosensor for pyruvate determination
15 times over one working day (Fig. 6, A); no
noticeable decrease in responses was observed.
The relative standard deviation of the biosen-
sor responses was 3.7%.
The operational stability is another very
important characteristic of the biosensor, which
determines the possibility of its continuous
application. To evaluate operational stability
of the PyrOx-based biosensor, three responses
to 500 µM pyruvate were measured once the
biosensor was prepared and after its storage
dry at + 4 °C before the next use. In a few
days, the biosensor was unfrozen, three re-
sponses were obtained, and the biosensor was
frozen again. In total, the test lasted for
14 days. The results obtained are presented in
Fig. 6, B. During the experiment, a slight de-
crease in responses was observed (final de-
crease was 18%). Thus, it is possible to pro-
ceed with the measurements for at least 14 days
or even more if an additional calibration is
carried out.
Selectivity of the biosensor
The mediator-free biosensor with high working
potential, used in the work, creates the prereq-
uisites for the oxidation of a number of electri-
cally active compounds other than tested,
which are present in biological samples. For
the improvement of the biosensor selectivity,
prior to the creation of bioselective elements
an additional layer of semi-permeable poly-
m-phenylenediamine (PPD) membrane was
deposited onto the electrode, which limits the
diffusion of interfering substances to its sur-
face. The procedure of formation of an addi-
tional polymer membrane consisted in elec-
tropolymerization of phenylenediamine mol-
ecules on the transducer surface. This tech-
nique has been developed in our laboratory
earlier [9]. It has been shown that after deposi-
tion of an additional PPD membrane the am-
perometric transducer was characterized by
high selectivity towards electrically active
substances such as ascorbic acid and cysteine.
A
B
Fig. 6. Reproducibility of biosensor response to pyruvate during continuous work (A) and operational stability of bio-
sensor during 14 days (B). Concentration of pyruvate was 500 µM, magnesium ions – 120 μM, TPP – 500 μM, PO4
3- –
20 mM. Measurements were carried out in 25 mM HEPES buffer, pH 7.4, at a constant potential of +0.6 V vs Ag/AgCl
reference electrode
22
D. V. Knyzhnykova, Ya. V. Topolnikova, I. S. Kucherenko et al.
It is highly probable that the developed
PyrOx-based biosensor will be integrated into
a multibiosensor (i.e. will function together
with other biosensors) to determine several key
metabolites (glucose, lactate, etc.). Therefore,
it was important to avoid any cross impact of
other substrates on the pyruvate analysis.
Hence, the biosensor sensitivity to possible
interfering substrates such as lactate, choline,
acetylcholine, glutamate and glucose was eval-
uated. It was found that the addition of other
than pyruvate substrates to the measuring cell
of the PyrOx-based biosensor had no impact
on its response, which proves high selectivity
of the biosensor to pyruvate.
Conclusions
The amperometric biosensor was developed
and optimized for the pyruvate determination.
Different variants of the PyrOx immobilization
were analyzed to create a bioselective element,
PyrOx photopolymerization in PVA-SbQ was
selected as optimal. The determined optimal
concentrations of cofactors and substrates for
the PyrOx-based biosensor were as follows:
magnesium – 120 μM, thiamine pyrophos-
phate – 500 μM, phosphoric acid ions –
20 mM. The developed biosensor had sensi ti-
vi ty to pyruvate of 31.06 nA/mM and a linear
working range of 0.01 to 5 mM. The biosensor
was shown to be highly selective towards elec-
trically active substances and other substrates,
which actually can be present in real samples.
Additionally, the biosensor is characterized by
high signal reproducibility over the working
day and operational stability during two weeks.
The developed biosensor is planned to be used
for the pyruvate measurement in biological
fluids.
Acknowledgements
This work was supported by the Program of
NAS of Ukraine “Smart sensor devices of a
new generation based on modern materials and
technologies“.
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Розробка амперометричного біосенсора на
основі піруватоксидази для визначення
пірувату
Д. В. Книжникова, Я. В. Топольнікова,
І. С. Кучеренко, О. О. Солдаткін
Мета. Розробка та оптимізація роботи амперометрич-
ного біосенсора для визначення пірувату. Методи.
Застосовано амперометричний біосенсор з іммобілізо-
ваною піруватоксидазою як біоселективний елемент та
платинові дискові електроди як перетворювачі.
Результати. Перевірено різні варіанти іммобілізації
піруватоксидази та обрано оптимальний для створення
біоселективного елементу біосенсора. Підібрано опти-
мальні концентрації кофакторів для найкращої роботи
біосенсора на основі піруватоксидази. Розроблений
біосенсор демонстрував високу чутливість до пірувату
та широкий лінійний діапазон роботи. Було показано
високу селективність запропонованого біосенсора від-
носно електроактивних речовин та інших субстратів,
що можуть бути присутніми в реальних зразках.
Біосенсор характеризується високою відтворюваністю
сигналу та операційною стабільністю протягом 2 тиж-
нів. Висновки. Розроблено високоселективний амперо-
метричний біосенсор для визначення пірувату у біо-
логічних зразках та досліджено його аналітичні харак-
теристики. В подальшому даний біосенсор може бути
використано для визначення пірувату у сироватці крові.
К л юч ов і с л ов а: піруват, піруватоксидаза, ампе-
рометричний біосенсор.
Разработка амперометрического биосенсора на
основе пируватоксидазы для определения
пирувата
Д. В. Книжникова, Я. В. Топольникова,
И. С. Кучеренко, А. А. Солдаткин
Цель. Разработка и оптимизация работы ампероме-
трического біосенсора для определения пирувата.
Методы. Использовано амперометрический біосенсор
с иммобилизованной пируватоксидазой как биоселек-
тивным элементом и платиновые дисковые электроды
как преобразователи. Результаты. Проверено разные
варианты иммобилизации пируватоксидазы и выбрано
оптимальный для создания биоселективного элемента
биосенсора. Подобрано оптимальные концентрации
кофакторов для наилучшей работы биосенсора на
основе пируватоксидазы. Разработанный биосенсор
демонстрировал высокую чувствительность к пирува-
ту и широкий линейный диапазон работы. Было пока-
зано хорошую селективность предложенного биосен-
сора относительно электроактивных веществ и других
субстратов, которые могут присутствовать в реальных
образцах. Биосенсор характеризуется высокой воспро-
изводимостью сигнала и операционной стабильностью
на протяжении двух недель. Выводы. Разработан
высокоселективный амперометрический биосенсор
для определения пирувата в биологических образцах
и исследованы его аналитические характеристики. В
дальнейшем данный биосенсор может быть использо-
ван для определения пирувата в сыворотке крови.
К л юч е в ы е с л ов а: пируват, пируватоксидаза,
амперометрический биосенсор.
Received 08.09.2017
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