Highly selective amperometric biosensor for uric acid determination in real samples
Aim. To develop an amperometric biosensor based on immobilized uricase (1.7.3.3) from Arthrobacter Globiformis and a platinum disk electrode for the detection of uric acid in biological fluids. Methods. To obtain a highly selective detection of the uric acid concentration, an additive semi-permeable...
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irk-123456789-1529172019-06-14T01:28:10Z Highly selective amperometric biosensor for uric acid determination in real samples Zinchenko, O.A. Shkotova, L.V. Kulynych, T.U. Zinkina, O.O. Soldatkin, A.P. Molecular and Cell Biotechnologies Aim. To develop an amperometric biosensor based on immobilized uricase (1.7.3.3) from Arthrobacter Globiformis and a platinum disk electrode for the detection of uric acid in biological fluids. Methods. To obtain a highly selective detection of the uric acid concentration, an additive semi-permeable polymer film was formed on the surface of a platinum disk electrode by electro-polymerisation of m-phenylene diamine. The enzymatic selective layer was formed on the poly-m-phenylene diamine membrane using uricase immobilized in BSA matrix by a non-toxic crosslinking agent – poly(ethylene glycol) diglycidyl ether (PEGDE). Results. An influence of possible interfering substances – ascorbic acid, cysteine, urea, glucose, glutamic acid and lactic acid – was studied. Almost no effect of these electrochemical compounds on the biosensor response was found, indicating that the selectivity of the developed biosensor is very high. The biosensor characteristics were determined: detection limit 0.001 mM (s/n = 3), linear working range 0.008–0.218 mM, sensitivity 165 μA·mM⁻¹ cm⁻². The biosensor stability and reproducibility were studied and shown. Conclusions. The developed biosensor was validated by comparing the results of the urine samples analysis provided with the biosensor and the spectrophotometric method (correlation coefficient r = 0.99).This biosensor is found to be promising method for uric acid detection in the real samples. Мета. Розробити амперометричний біосенсор на основі іммобілізованої урікази (1.7.3.3) з Arthrobacter Globiformis і платинового дискового електрода для визначення сечової кислоти в біологічних рідинах. Методи. Для досягнення високоселективного визначення концентрації сечової кислоти на поверхні платинового дискового електрода сфо-рмована додаткова напівпроникна мембрана шляхом електрополімерізаціі м-фенілендіаміна. Ферментний селективний шар сформований на полі-м-фенілендіаміновій мембрані з використанням урікази, що іммобілізована в матриці БСА, в якості зшиваючого агента використовували нетоксичний поліетиленгліколь дигліцидиловий естер (ПЕГДЕ). Результати. Досліджено вплив інтерферуючих речовин: аскорбінової кислоти, цистеїну, сечовини, глюкози, глутамінової кислоти, молочної кислоти на активність розробленого біосенсору і показана відсутність впливу цих електрохімічно активних речовин на відгук біосенсора, що свідчить про дуже високу селективність розробленого біосенсора. Визначені наступні характеристики біосенсора: мінімальна концентрація, що визначалась 0.001 мM (s/n = 3), робочий лінійний діапазон 0.008–0.218 мM, чутливість 165 мкА∙мМ⁻¹ см⁻². Також досліджені і пока-зані операційна стабільність біосенсора і його стабільність при зберіганні. Висновки. Апробація розробленого біо-сенсора при аналізі реальних зразків сечі показала хорошу кореляцію даних із класичним спектрофотометричним методом (коефіцієнт кореляції r = 0.99). Таким чином, даний біосенсор є перспективним методом і може бути застосований в медичній діагностиці для визначення сечової кислоти в реальних зразках. Цель. Разработать амперометрический биосенсор на основе иммобилизированной уриказы (1.7.3.3) из Arthrobacter Globiformis и платинового дискового электрода для определения мочевой кислоты в биологических жидкостях. Методы. Для достижения высокоселективного определения концентрации мочевой кислоты на поверхности платинового дискового электрода была сформирована дополнительная полупроницаемая мембрана путём электрополимеризации м-фенилендиамина. Ферментный селективный слой сформирован на поли-м-фенилендиаминовой мембране с использованием уриказы, иммобилизированной в матрице БСА, в качестве сшивающего агента использовали нетоксичный диглицидиловый эфир полиэтиленгликоля (ПЭГДЭ). Результаты. Исследовано влияние интерферирующих веществ: аскорбиновой кислоты, цистеина, мочевины, глюкозы, глутаминовой кислоты, мо-лочной кислоты на активность разработанного биосенсора и показано отсутствие влияния этих электрохимически активных веществ на отклик биосенсора, что свидетельствует об очень высокой селективности разработанного биосенсора. Определены следующие характеристики биосенсора: граничная определяемая концентрация 0.001 мM (s/n = 3), рабочий линейный диапазон 0.008–0.218 мM, чувствительность 165 мкА•мМ⁻¹ см⁻². Также исследованы и продемонстрированы операционная стабильность биосенсора и его стабильность при хранении. Выводы. Апробация разработанного биосенсора при анализе реальных образцов мочи показала хорошую корреляцию данных с классическим спектрофотометрическим методом (коэффициент корреляции r = 0.99). Таким образом, данный биосенсор является перспективным методом и может быть использован в медицинской диагностике для определения мочевой кислоты в реальных образцах. 2017 Article Highly selective amperometric biosensor for uric acid determination in real samples / O.A. Zinchenko, L.V. Shkotova, T.U. Kulynych, O.O. Zinkina, A.P. Soldatkin // Вiopolymers and Cell. — 2017. — Т. 33, № 2. — С. 124-134. — Бібліогр.: 24 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00094A http://dspace.nbuv.gov.ua/handle/123456789/152917 543.553+544.722+543.64+543.94 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Molecular and Cell Biotechnologies Molecular and Cell Biotechnologies |
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Molecular and Cell Biotechnologies Molecular and Cell Biotechnologies Zinchenko, O.A. Shkotova, L.V. Kulynych, T.U. Zinkina, O.O. Soldatkin, A.P. Highly selective amperometric biosensor for uric acid determination in real samples Вiopolymers and Cell |
| description |
Aim. To develop an amperometric biosensor based on immobilized uricase (1.7.3.3) from Arthrobacter Globiformis and a platinum disk electrode for the detection of uric acid in biological fluids. Methods. To obtain a highly selective detection of the uric acid concentration, an additive semi-permeable polymer film was formed on the surface of a platinum disk electrode by electro-polymerisation of m-phenylene diamine. The enzymatic selective layer was formed on the poly-m-phenylene diamine membrane using uricase immobilized in BSA matrix by a non-toxic crosslinking agent – poly(ethylene glycol) diglycidyl ether (PEGDE). Results. An influence of possible interfering substances – ascorbic acid, cysteine, urea, glucose, glutamic acid and lactic acid – was studied. Almost no effect of these electrochemical compounds on the biosensor response was found, indicating that the selectivity of the developed biosensor is very high. The biosensor characteristics were determined: detection limit 0.001 mM (s/n = 3), linear working range 0.008–0.218 mM, sensitivity 165 μA·mM⁻¹ cm⁻². The biosensor stability and reproducibility were studied and shown. Conclusions. The developed biosensor was validated by comparing the results of the urine samples analysis provided with the biosensor and the spectrophotometric method (correlation coefficient r = 0.99).This biosensor is found to be promising method for uric acid detection in the real samples. |
| format |
Article |
| author |
Zinchenko, O.A. Shkotova, L.V. Kulynych, T.U. Zinkina, O.O. Soldatkin, A.P. |
| author_facet |
Zinchenko, O.A. Shkotova, L.V. Kulynych, T.U. Zinkina, O.O. Soldatkin, A.P. |
| author_sort |
Zinchenko, O.A. |
| title |
Highly selective amperometric biosensor for uric acid determination in real samples |
| title_short |
Highly selective amperometric biosensor for uric acid determination in real samples |
| title_full |
Highly selective amperometric biosensor for uric acid determination in real samples |
| title_fullStr |
Highly selective amperometric biosensor for uric acid determination in real samples |
| title_full_unstemmed |
Highly selective amperometric biosensor for uric acid determination in real samples |
| title_sort |
highly selective amperometric biosensor for uric acid determination in real samples |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2017 |
| topic_facet |
Molecular and Cell Biotechnologies |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/152917 |
| citation_txt |
Highly selective amperometric biosensor for uric acid determination in real samples / O.A. Zinchenko, L.V. Shkotova, T.U. Kulynych, O.O. Zinkina, A.P. Soldatkin // Вiopolymers and Cell. — 2017. — Т. 33, № 2. — С. 124-134. — Бібліогр.: 24 назв. — англ. |
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Вiopolymers and Cell |
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124
O. A. Zinchenko, L. V. Shkotova, T. U. Kulynych
© 2017 O. A. Zinchenko et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio-
polymers 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 543.553+544.722+543.64+543.94
Highly selective amperometric biosensor for uric acid determination
in real samples
O. A. Zinchenko1, L. V. Shkotova1, T. U. Kulynych2, O. O. Zinkina3,
A. P. Soldatkin
1 Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
2 Taras Shevchenko National University of Kyiv
64, Volodymyrska Str., Kyiv, Ukraine, 01601
3 Institute of High Technologies, Taras Shevchenko National University of Kyiv
2, korp.5, Pr. Akademika Hlushkova, Kyiv, Ukraine, 03022
helen_nazarenko@yahoo.com
Aim. To develop an amperometric biosensor based on immobilized uricase (1.7.3.3) from
Arthrobacter Globiformis and a platinum disk electrode for the detection of uric acid in bio-
logical fluids. Methods. To obtain a highly selective detection of the uric acid concentration,
an additive semi-permeable polymer film was formed on the surface of a platinum disk elec-
trode by electro-polymerisation of m-phenylene diamine. The enzymatic selective layer was
formed on the poly-m-phenylene diamine membrane using uricase immobilized in BSA matrix
by a non-toxic crosslinking agent – poly(ethylene glycol) diglycidyl ether (PEGDE). Results.
An influence of possible interfering substances – ascorbic acid, cysteine, urea, glucose, glu-
tamic acid and lactic acid – was studied. Almost no effect of these electrochemical compounds
on the biosensor re-sponse was found, indicating that the selectivity of the developed biosen-
sor is very high. The bio-sensor characteristics were determined: detection limit 0.001 mM
(s/n = 3), linear working range 0.008–0.218 mM, sensitivity 165 µA•mM–1 cm–2. The biosen-
sor stabi-li-ty and reproducibility were studied and shown. Conclusions. The developed bio-
sensor was validated by comparing the results of the urine samples analysis provided with the
biosensor and the spectrophotometric method (correlation coefficient r = 0.99).This biosensor
is found to be promising method for uric acid detection in the real samples.
K e y w o r d s: uricase; amperometric biosensor; uric acid; m-phenylene diamine; poly(ethylene
glycol) diglycidyl ether.
Introduction
Uric acid (2,4,6 – trihydroxypurine) is an end
product of the purine metabolism in humans.
A number of diseases, sometimes very serious,
are known to be caused by the violations of
Molecular and Cell
Biotechnologies
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2017. Vol. 33. N 2. P 124–134
doi: http://dx.doi.org/10.7124/bc.00094A
125
Highly selective amperometric biosensor for uric acid determination in real samples
purine metabolism, generally declared as a
significant increase of uric acid in the blood.
It can result for instance, in the development
of hyperuricemia (Lesch–Nyhan syndrome) [1]
and gout [2–3]. According to the epidemio-
logical studies, on average 0.01–0.37 % of
adults worldwide suffer from gout, caused by
the kidney failure, as well as hematological
diseases, myocardial infarction, and heart fail-
ure [2]. Several medical investigations dem-
onstrated that an increased level of uric acid
in human serum was a risk factor for cardio-
vascular disease [4]. The normal level of uric
acid ranges from 240 to 520 µM in blood se-
rum and 1.4 to 4.4 mM in urine [5], increasing
by 3–4 times under at pathology.
Therefore, the determination of uric acid
concentration in biological fluids is important
in the laboratory practice. A number of meth-
ods are used for the analysis – chemical [6],
enzymatic-colorimetric [7], chemiluminescent
[8–9], fluorescent [10], voltammetric-colori-
metric [11], enzymatic-spectrophotometric
[12], mass spectrometric [13], high perfor-
mance liquid chromatography [14], capillary
electrophoresis, and amperometry [15].
However, despite the noticeable diagnostic
importance of the quantity of uric acid con-
centration, its analysis is not widely used in
clinical practice because of the low specificity
and complex procedure of the existing me-
thods.
The Biosensors can be considered as a good
alternative method of the uric acid detec-
tion [16].
In this work an amperometric biosenor for
the determination of uric acid has been deve-
lo ped using a promising method of the uricase
immobilization with poly(ethylene glycol) di-
glycidyl ether (PEGDE) on the transducer
surfaces. PEGDE, an important component of
the redox hydrogels, is broadly used in the
commercial devices. It is a non-toxic chemical,
which is utilized for the enzyme fixation. It
contains two epoxy groups able to react with
the amino-, hydroxyl- and carboxyl groups of
enzymes [17]. Under the influence of tem-
perature PEGDE reacts with amino groups of
the enzyme at higher rate and forms a matrix
for uricase immobilization on the electrode
surface, which is very similar to the matrix
formed with glutaraldehyde.
Previously it was shown [18] that the im-
mobilization with PEGDE has a slight effect
on the enzyme catalytic parameters and at the
same time the immobilized enzyme is stable
and selective.
Materials and Methods
Materials
Uricase (1.7.3.3.), 15-30 U/mg of protein from
Artrobacter globiformis, production of
“Sigma” (Germany); uric acid (99% purity),
production of “Sigma” (Hungary); 99% etha-
nol, production of “Fluka” (Germany); bovine
serum albumin (BSA) (fraction V), production
of “Sigma” (Germany); poly(ethylene glycol)
diglycidyl ether (Mn 500), production of
“Sigma” (Japan); 1,3 phenylenediamine, pro-
duction of “Sigma-Aldrich Chemie GmbH;
borate buffer (Na2B4O7x10H2O-HCl), pH 8.5,
Na2HPO4·7H2O and KH2PO4·H2O, production
of “Helicoη” (Russia); 3 % solution of hydro-
gen peroxide (Teteriv, Kiev region, Ukraine);
Na2SO4·10H2O, production of Mikhailovsky
chemical reagent factory (Russia). Polymix
buffer solution (NaH2PO4, Na2B4O7, Tris-HCl,
126
O. A. Zinchenko, L. V. Shkotova, T. U. Kulynych et al.
KCl, NaON and citric acid) production of
“Helicoη” (Russia). All chemicals used were
of analytical reagent grade.
A scheme of measuring setup
Amperometric measurements were carried out
in the 2 ml electrochemical cell using a poten-
tiostat/galvanostat PalmSens (Netherlands pro-
duction) controlled by the PalmSens PC pro-
gramme (Fig. 1). The three-electrode circuit
consisted of platinum working, platinum aux-
iliary and Ag/AgCl reference electrodes [19].
The amperometric platinum transducers
were studied with regards to the signal repro-
ducibility and reliability by cyclic voltam-
perometry in the potential range of 0 to +1.0 V
versus the Ag/AgCl reference electrode
(a speed of potential involute was 0.05 V/s).
The experiments were carried out in 5 mM
borate buffer, pH 8.5.
Procedure of deposition of additional
poly-m-phenylene diamine membranes
on the surface of amperometric platinum
electrodes
The unmodified amperometric platinum trans-
ducers are characterized by their sensitivity to
a variety of electroactive substances. To im-
prove the transducer selectivity two appro-
aches were used: reduction of working poten-
tial and deposition of semi-permeable mem-
branes on the transducer surface. Such mem-
branes are permeable for small compounds,
such as hydrogen peroxide, and are a barrier
for diffusion of bigger compounds to the elec-
trodes. For the membrane deposition, a bare
transducer was immersed in 1 mM solution of
m-phenylene diamine (m-PD), afterwards five
cyclic voltammograms were obtained (if more
than five cycles of polymerization are per-
formed the membrane becomes too dense,
which results in the significantly reduced sen-
sitivity of the transducer to hydrogen pero-
xide). m-Phenylene diamine was dissolved in
10 mM potassium-phosphate buffer, pH 7.2.
The initial potential was 0 V, the end potential
+0.9 V, the rate of potential change was 0.2 V/s
in the presence of 5 mM m-phenylene diamine.
Before the subsequent deposition of the biose-
lective membrane, the surface of poly-m -PD-
modified transducer was thoroughly washed
with distilled water. The technique of deposi-
tion of the poly-m-phenylene diamine mem-
brane was adapted from [20].
The cyclic voltammogram of poly-m-PD
electropolymerisation demonstrates a decrease
of the peak values in each next cycle caused
by the formation of a polymer film on the
working electrode surface.
Preparation of bioselective membrane
To prepare an uricase-based bioselective ele-
ment the required amounts of the enzyme and
BSA were dissolved in 20 mM phosphate buf-
fer, pH 8.5, with addition of glycerol for the
enzyme stabilization and prevention of early
drying of the solution on the transducer sur-
face. The obtained solution was mixed with
PEGDE just before the membrane preparation.
The final mixture contained 50 mg/ml uricase,
50 mg/ml BSA, 5 % of glycerol and 40 mg/
ml PEGDE in 20 mM phosphate buffer,
pH 8.5. The prepared mixture was immedi-
ately deposited on the sensitive surface of
platinum disk electrode; 0.1 µl was needed to
cover it completely. For immobilization the
electrode was placed in the oven at 55 oC for
2h (see Fig. 1).
127
Highly selective amperometric biosensor for uric acid determination in real samples
Preparation of uric acid solution.
5 mM solution of uric acid in the 5 mM borate
buffer, pH 8.5, was prepared just before the mea-
surement. For complete dissolution of uric acid
the solution was heated to 50 oС. pH of uric acid
solution was adjusted to pH 8.5 with 1M NaOH.
Procedure of measuring substrates
in model solutions
Biosensor measurements of the uric acid con-
centrations were carried out at room tempera-
ture in an open electrochemical cell filled with
working buffer (mostly 5 mM borate buffer,
pH 8.5) at vigorous stirring. Before addition
of uric acid, the transducers were kept in the
working buffer solution until the stable signal
(base line) was obtained. The substrate con-
centration in the electrochemical cell was
changed by adding aliquots of the substrate
stock solutions. The experiments were carried
out in at least three replications.
Statistics
Statistical package Microsoft Excel 10 was
used for statistical analysis of the results, the
average values and standard deviations were
calculated; the results were considered as reli-
able at p < 0.05.
Results and Discussion
Transducer optimization
The biosensor selectivity depends on two
major factors – selectivity of an enzyme (an
enzyme-substrate reaction) and selectivity of
a transducer (influence of electrochemical
interferents). To minimize the effect of in-
terfering substances, it was created a semi-
permeable selective membrane, which pre-
vents an access of undesirable active com-
pounds to the transducer surface. An addi-
tional polymeric membrane obtained by
electrochemical oxidation of water-soluble
monomer phenylene diamine was used for
the purpose. The best sensitivity and selectiv-
ity were reported in case of meta-phenylene
diamine, whereas ortho-phenylene diamine
provided high sensitivity but low specificity,
para-phenylene diamine - low sensitivity and
selectivity [20].
Fig. 1. Scheme of the enzyme immobilization by PEGDE [18].
128
O. A. Zinchenko, L. V. Shkotova, T. U. Kulynych et al.
Fig. 2. Comparison of responses of plat-
inum electrodes (A) (bare and covered
with poly-m-phenylene diamine layer)
to uric acid, hydrogen peroxide and po-
tentially possible interfering substances.
Sensitivity to hydrogen peroxide of plat-
inum electrodes: bare (B(1)) and modi-
fied with electropolymerised layer of
poly-m-phenylene diamine (B(2)). Mea-
surement conditions: 5 mM borate buf-
fer (pH 8.5), at potential of +0.4 V ver-
sus reference electrode.
The selectivity of bare electrodes and elec-
trodes coated with poly-m-phenylene diamine
to possible interferents was studied via as-
sessing their sensitivity response to uric acid,
ascorbic acid, cysteine, glutamic acid, urea,
lactate, glucose. As shown in Fig.2 (A), bare
electrodes responded to a wide range of in-
terfering substances, whereas the polymer-
coated electrodes almost did not react to
them.
A
B
129
Highly selective amperometric biosensor for uric acid determination in real samples
Hence, in practical measurements, the min-
imal sensitivity of transducer to possible inter-
fering substances can be neglected and this
design of transducers can be successfully used
for the further development of the biosensor
for uric acid analysis.
A comparison of the sensitivity of bare and
modified electrodes to hydrogen peroxide
showed that after deposition of a poly-m-phen-
ylenediamine layer onto the platinum electrode
surface, the sensitivity to H2O2 decreased
slightly (see Fig.2 (B)).
Biosensor
All known methods for the enzymatic deter-
mination of uric acid using uricase are based
on the enzymatic oxidation of uric acid with
production of allantoin, hydrogen peroxide and
carbon dioxide:
Uricase
Uric acid +H2O + O2 →
→ Allantoin + H2O2 + CO2
Decomposition of electrically active sub-
stance hydrogen peroxide results in generation
of electrons, which can be registered by am-
perometric transducer
H2O2 → 2H+ + 2e-
In the work presented, we used uricase for
creation of the biosensor and the enzyme was
immobilized on the transducer surface by poly
(ethylene glycol) diglycidyl ether crosslinking.
The measurements were generally carried
out at room temperature in an intensively
stirred electrochemical open cell system. The
typical dependence of the biosensor response
on the uric acid concentration (calibration
curve) is presented in Fig. 3.
As can be seen from Fig 3 the apparent
Michaelis-Menten constant (Km) is 0.06 mM
and Imax = 22 nA for uricase immobilized in
a bioselective membrane. A linear dependence
of the biosensor response was observed in the
range of uric acid concentration 0.008 –
0.218 mM (s/n =3), with the sensor sensitivity
of 165 µA·mM–1·сm–2 (the calibration curve
Fig. 3. Calibration curves for the uric
acid sensitive biosensor. Measurement
conditions: 5 mM borate buffer (pH 8.5),
at potential of +0.4 V versus reference
electrode.
130
O. A. Zinchenko, L. V. Shkotova, T. U. Kulynych et al.
presented in linear coordinates). Under the
conditions described, the time of biosensor
response was about 5–10 s. The presentation
of the biosensor calibration curve in semi-
logarithmic coordinates gives the possibility
to extend the linear dynamic range and to shift
the range of uric acid detection to higher con-
centration.
A low value of Michaelis–Menten constant
(0.06 mM) indicates that immobilized uricase
in the BSA-PEGDE layer has high affinity to
uric acid. The detection limit of the uricase
biosensor was estimated as 0.001 mM (s/n =3).
Since the biosensor working characteristics
strongly depend on the experimental condi-
tions, it was important to examine an effect of
ionic strength and pH on the biosensor re-
sponse. The dependence of amperometric bio-
sensor response on the NaCl concentration is
shown in Fig.4(A).
An analysis of the impact of NaCl concen-
trations revealed a little effect on the response
of amperometric uricase-based biosensor.
Notably, it is typical for most enzyme ampero-
metric biosensors [21].
As known, each enzyme has a specific
working optimum pH value. For some enzyme
it can be changed after immobilization, shifting
into alkaline or acid region. This phenomenon
can be explained in the first place by the
change in conformation of immobilized en-
zyme and redistribution of charges within the
enzyme-BSA-PEGDE system. The pH-depen-
dence of the sensor response was investigated
for 3, 8 and 18 µM uric acid over the pH range
of 6.0–11.0 in the universal buffer solu-
tion [22]. The experimental results show (see
Fig.4 (B)) an atypical picture of dependence
of the biosensor responses on pH; it means that
the activity of immobilized uricase is weakly
A
B
Fig. 4. Dependence of the response of amperometric uricase-based biosensor on the concentration of NaCl in a buffer
solution (A). Measurement conditions: 5 mM borate buffer, pH 8.5. Dependence of the response of amperometric bio-
sensors based on immobilized uricase on pH of polymix buffer solution (B) (2.5 mM NaH2PO4, 2,5 mM Na2B4O7,
2,5 mM Tris-HCl, 2,5 mM KCl, 2,5 mM NaON and 2,5 mM citric acid). Measurement was carried out at potential of
+ 0.4 V versus reference electrode.
131
Highly selective amperometric biosensor for uric acid determination in real samples
dependent on the pH changes in this range.
The form of curves can be explained by an
increase in the immobilized enzyme stability
and/or by the limitation of substrate diffusion
in a bioselective membrane [23]. The highest
activity was observed at pH 8.5, which was
chosen as optimal for further work.
The response reproducibility and opera-
tional stability, the most important biosensor
characteristics, were studied. The biosensor
responses to different concentrations of uric
acid were measured over one working day with
30-min intervals, the biosensor with immobi-
lized uricase being kept between measure-
ments in buffer solution at room temperature.
The biosensor was characterized by high re-
producibility (relative standard deviation of
the signals did not exceed 5 %) and high op-
erational stability (the bioselective element did
not lose its activity during 5–6 h).
To study long-term stability of the biosensor
developed, the responses to the same concen-
tration of substrate were evaluated with 100-
hour intervals between subsequent measure-
ments (data not presented). During the first
100 hours of storage, the biosensors lost 30 %
of the initial sensitivity. In 400 hours, the bio-
sensors still exhibited approximately 50 % of
their initial sensitivity. The loss of activity is
probably associated with the relatively low
stability of uricase.
Analysis of real samples
An efficiency of the developed biosensor for
in the analysis of real samples was evaluated
by comparing the values of uric acid concen-
tration in the urine samples of 15 volunteers
measured by the presented biosensor method
(axis Y) with the results obtained by spectro-
photometric method [24] (axis X) (Fig. 5).
A high correlation was demonstrated (correla-
tion coefficient r = 0.99).
Conclusion
The electrochemical biosensor based on the
poly(m-phenylenediamine) modified platinum
disk electrode as a transducer and immobilized
uricase from Arthobacter globiformis as a sen-
sitive element has been developed for detection
of uric acid in the presence of electrochemical
compounds. It provides a sensitive, selective
and rapid methods of uric cid analysis.
The amperometric biosensor for detection
of uric acid in biological fluids was developed
using uricase immobilization by a non-toxic
crosslinking agent – poly(ethylene glycol)di-
glycidyl ether.
The main characteristics of the developed bio-
sensor were estimated. The biosensor demon-
strated fast response (5s), high sensitivity and
selectivity, linear working range of 0.01 – 0.22 mM
with detection limit of 0.001 mM (s/n =3).
Fig. 5. Correlation between the values of uric acid concen-
trations in urine determined by the developed biosensor
and the hospital standard spectrophotometric me thods.
132
O. A. Zinchenko, L. V. Shkotova, T. U. Kulynych et al.
The biosensor was tested when analyzing
the urine samples of 15 volunteers. The results
correlated with those obtained by the standard
spectrophotometric method. Thus, the develo-
ped biosensor can be applied for monitoring
the level of uric acid in urine at norm and
pathology during the medical examination of
population.
Acknowledgments
The authors gratefully acknowledge the
financial support of this study by the National
Academy of Sciences of Ukraine in the frame
of Scientific and Technical Program “Sensor
systems for medico-ecological and industrial-
technological requirement: metrological sup-
port and experimental operation”.
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Високоселективний амперометричний
біосенсор для визначення сечової кислоти
в реальних зразках
О. А. Зінченко, Л. В. Шкотова, Т. Ю. Кулинич,
О. О. Зінкіна, О. П. Солдаткін
Мета. Розробити амперометричний біосенсор на осно-
ві іммобілізованої урікази (1.7.3.3) з Arthrobacter
Globiformis і платинового дискового електрода для
визначення сечової кислоти в біологічних рідинах.
Методи. Для досягнення високоселективного визна-
чення концентрації сечової кислоти на поверхні пла-
тинового дискового електрода сформована додаткова
напівпроникна мембрана шляхом електрополімерізаціі
м-фенілендіаміна. Ферментний селективний шар сфор-
мований на полі-м-фенілендіаміновій мембрані з ви-
користанням урікази, що іммобілізована в матриці
БСА, в якості зшиваючого агента використовували
нетоксичний поліетиленгліколь дигліцидиловий естер
(ПЕГДЕ). Результати. Досліджено вплив інтерферу-
ючих речовин: аскорбінової кислоти, цистеїну, сечо-
вини, глюкози, глутамінової кислоти, молочної кисло-
ти на активність розробленого біосенсору і показана
відсутність впливу цих електрохімічно активних ре-
човин на відгук біосенсора, що свідчить про дуже
високу селективність розробленого біосенсора.
Визначені наступні характеристики біосенсора: міні-
мальна концентрація, що визначалась 0.001 мM
(s/n = 3), робочий лінійний діапазон 0.008–0.218 мM,
чутливість 165 мкА∙мМ–1 см–2. Також досліджені і по-
казані операційна стабільність біосенсора і його ста-
більність при зберіганні. Висновки. Апробація роз-
робленого біосенсора при аналізі реальних зразків сечі
показала хорошу кореляцію даних із класичним спек-
трофотометричним методом (коефіцієнт кореляції r =
0.99). Таким чином, даний біосенсор є перспективним
методом і може бути застосований в медичній діа-
гностиці для визначення сечової кислоти в реальних
зразках.
К л юч ов і с л ов а: уріказа; амперометричний біо-
сенсор; сечова кислота; м-фенілендіамін; поліетилен-
гліколь дигліцидиловий естер.
Высокоселективный амперометрический
биосенсор для определения мочевой кислоты
в реальных образцах
Е. А. Зинченко, Л. В. Шкотова, Т. Ю. Кулинич,
О. А. Зинкина, А. П. Солдаткин
Цель. Разработать амперометрический биосенсор на
основе иммобилизированной уриказы (1.7.3.3) из
Arthrobacter Globiformis и платинового дискового элек-
трода для определения мочевой кислоты в биологиче-
ских жидкостях. Методы. Для достижения высокосе-
лективного определения концентрации мочевой кис-
лоты на поверхности платинового дискового электро-
да была сформирована дополнительная полупроница-
емая мембрана путём электрополимеризации
м-фенилендиамина. Ферментный селективный слой
сформирован на поли-м-фенилендиаминовой мембра-
134
O. A. Zinchenko, L. V. Shkotova, T. U. Kulynych et al.
не с использованием уриказы, иммобилизированной
в матрице БСА, в качестве сшивающего агента исполь-
зовали нетоксичный диглицидиловый эфир полиэти-
ленгликоля (ПЭГДЭ). Результаты. Исследовано вли-
яние интерферирующих веществ: аскорбиновой кис-
лоты, цистеина, мочевины, глюкозы, глутаминовой
кислоты, молочной кислоты на активность разрабо-
танного биосенсора и показано отсутствие влияния
этих электрохимически активных веществ на отклик
биосенсора, что свидетельствует об очень высокой
селективности разработанного биосенсора. Опре де-
лены следующие характеристики биосенсора: гранич-
ная определяемая концентрация 0.001 мM (s/n = 3),
рабочий линейный диапазон 0.008–0.218 мM, чувстви-
тельность 165 мкА•мМ–1 см–2. Также исследованы и
продемонстрированы операционная стабильность
биосенсора и его стабильность при хранении. Выводы.
Апробация разработанного биосенсора при анализе
реальных образцов мочи показала хорошую корреля-
цию данных с классическим спектрофотометрическим
методом (коэффициент корреляции r = 0.99). Таким
образом, данный биосенсор является перспективным
методом и может быть использован в медицинской
диагностике для определения мочевой кислоты в ре-
альных образцах.
К л юч е в ы е с л ов а: уриказа; амперометрический
биосенсор; мочевая кислота; м-фенилендиамин; поли-
этиленгликоль диглицидиловый эфир.
Received 15.01.2017
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