Conductometric urease microbiosensor based on thin-film interdigitated electrodes for urea determination
The characteristics of conductometric urease microbiosensor based on thin-film inter-digitated electrodes for urea determination have been studied. Urease has been immobilized by cross-linking with bovine serumalbumin using glutaraldehyde. The resulting conductivity changes are produced by enzymatic...
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1996
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| Cite this: | Conductometric urease microbiosensor based on thin-film interdigitated electrodes for urea determination / S.V. Dzyadevich, G.A Zhylyak., A.P. Soldatkin, A.V. El'skaya // Биополимеры и клетка. — 1996. — Т. 12, № 1. — С. 53-57. — Бібліогр.: 9 назв. — англ. |
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Dzyadevich, S.V. Zhylyak, G.A. Soldatkin, A.P. El'skaya, A.V. 2019-06-14T16:16:19Z 2019-06-14T16:16:19Z 1996 Conductometric urease microbiosensor based on thin-film interdigitated electrodes for urea determination / S.V. Dzyadevich, G.A Zhylyak., A.P. Soldatkin, A.V. El'skaya // Биополимеры и клетка. — 1996. — Т. 12, № 1. — С. 53-57. — Бібліогр.: 9 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000412 https://nasplib.isofts.kiev.ua/handle/123456789/153794 577.15+573.6 The characteristics of conductometric urease microbiosensor based on thin-film inter-digitated electrodes for urea determination have been studied. Urease has been immobilized by cross-linking with bovine serumalbumin using glutaraldehyde. The resulting conductivity changes are produced by enzymatically catalyzed hydrolysis of urea. This process for both immobilized enzyme and soluble one is described by the classic laws of enzymatic kinetics. The influence of ionic strength, pH and buffer capacity of the samples on the biosensor response has been thoroughly tested. The results have been discussed concerning urea concentration analysis in human blood. Вивчено характеристики кондуктометричного уреазного біосенсора для визначення сечовини на основі тонкоплівкових гребінчастих електродів. Уреазу іммобілізовали ковалентною зшивкою з сироватковим альбуміном бика за допомогою глутарового альдегіду. Результуючі зміни провідності викликано ферментативним гідролізом сечовини. Згаданий процес як для іммобілізова ного, так і для вільного ферменту описується класичними законами ферментативної кінетики. Досліджено вплив на величину відгуку іонної сили, рН та буферної ємності розчину. Розглянуто питання стосовно визначення концентрації сечовини у крові людини. Изучены характеристики кондуктометрического уреазного микробиосенсора для определения мочевины на основе тонкопленочных гребенчатых электродов. Уреазу иммобилизовали ковалентной сшивкой с бычьим сывороточным альбумином с помощью глутарового альдегида. Результирующее изменение проводимости вызвано ферментативным гидролизом мочевины. Этот процесс как для иммобилизованного, так и для свободного фермента описывается классическими законами ферментативной кинетики. Исследовано влияние на величину отклика ионной силы, рН и буферной емкости раствора Рассмотрены вопросы, связанные с определением концентра ции мочевины в крови человека. A part of this work was supported by a grant from State Committee of Ukraine on Science and Technology (grant No. 5.3/21). en Інститут молекулярної біології і генетики НАН України Биополимеры и клетка Conductometric urease microbiosensor based on thin-film interdigitated electrodes for urea determination Кондуктометричний уреазний мікробіосенсор для визначення сечовини на основі тонкоплівкових гребінчастих електродів Кондуктометрический уреазный микробиосенсор для определения мочевины на основе тонкопленочных гребенчатых электродов Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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| title |
Conductometric urease microbiosensor based on thin-film interdigitated electrodes for urea determination |
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Conductometric urease microbiosensor based on thin-film interdigitated electrodes for urea determination Dzyadevich, S.V. Zhylyak, G.A. Soldatkin, A.P. El'skaya, A.V. |
| title_short |
Conductometric urease microbiosensor based on thin-film interdigitated electrodes for urea determination |
| title_full |
Conductometric urease microbiosensor based on thin-film interdigitated electrodes for urea determination |
| title_fullStr |
Conductometric urease microbiosensor based on thin-film interdigitated electrodes for urea determination |
| title_full_unstemmed |
Conductometric urease microbiosensor based on thin-film interdigitated electrodes for urea determination |
| title_sort |
conductometric urease microbiosensor based on thin-film interdigitated electrodes for urea determination |
| author |
Dzyadevich, S.V. Zhylyak, G.A. Soldatkin, A.P. El'skaya, A.V. |
| author_facet |
Dzyadevich, S.V. Zhylyak, G.A. Soldatkin, A.P. El'skaya, A.V. |
| publishDate |
1996 |
| language |
English |
| container_title |
Биополимеры и клетка |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Кондуктометричний уреазний мікробіосенсор для визначення сечовини на основі тонкоплівкових гребінчастих електродів Кондуктометрический уреазный микробиосенсор для определения мочевины на основе тонкопленочных гребенчатых электродов |
| description |
The characteristics of conductometric urease microbiosensor based on thin-film inter-digitated electrodes for urea determination have been studied. Urease has been immobilized by cross-linking with bovine serumalbumin using glutaraldehyde. The resulting conductivity changes are produced by enzymatically catalyzed hydrolysis of urea. This process for both immobilized enzyme and soluble one is described by the classic laws of enzymatic kinetics. The influence of ionic strength, pH and buffer capacity of the samples on the biosensor response has been thoroughly tested. The results have been discussed concerning urea concentration analysis in human blood.
Вивчено характеристики кондуктометричного уреазного біосенсора для визначення сечовини на основі тонкоплівкових гребінчастих електродів. Уреазу іммобілізовали ковалентною зшивкою з сироватковим альбуміном бика за допомогою глутарового альдегіду. Результуючі зміни провідності викликано ферментативним гідролізом сечовини. Згаданий процес як для іммобілізова ного, так і для вільного ферменту описується класичними законами ферментативної кінетики. Досліджено вплив на величину відгуку іонної сили, рН та буферної ємності розчину. Розглянуто питання стосовно визначення концентрації сечовини у крові людини.
Изучены характеристики кондуктометрического уреазного микробиосенсора для определения мочевины на основе тонкопленочных гребенчатых электродов. Уреазу иммобилизовали ковалентной сшивкой с бычьим сывороточным альбумином с помощью глутарового альдегида. Результирующее изменение проводимости вызвано ферментативным гидролизом мочевины. Этот процесс как для иммобилизованного, так и для свободного фермента описывается классическими законами ферментативной кинетики. Исследовано влияние на величину отклика ионной силы, рН и буферной емкости раствора Рассмотрены вопросы, связанные с определением концентра ции мочевины в крови человека.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/153794 |
| citation_txt |
Conductometric urease microbiosensor based on thin-film interdigitated electrodes for urea determination / S.V. Dzyadevich, G.A Zhylyak., A.P. Soldatkin, A.V. El'skaya // Биополимеры и клетка. — 1996. — Т. 12, № 1. — С. 53-57. — Бібліогр.: 9 назв. — англ. |
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I S S N 0233-7657. Биополимеры и клетка. 1996. Т. 12. № 1
53
Conductometric urease microbiosensor based on thin-film
interdigitated electrodes for urea determination
Sergey V. Dzyadevich*, Gleb A. Zhylyak, Alexey P. Soldatkin,
Anna V. El'skaya
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine,
150 Zabolotnogo str., Kyiv, 252143, Ukraine
he characteristics of conductometric urease microbiosensor based on thin-film inter
digitated electrodes for urea determination have been studied. Urease has been immobi
lized by cross-linking with bovine serum albumin using glutaraldehyde. The resulting con
ductivity changes are produced by enzymatically catalyzed hydrolysis of urea. This process
for both immobilized enzyme and soluble one is described by the classic laws of enzymatic
kinetics. The influence of ionic strength, pH and buffer capacity of the samples on the
biosensor response has been thoroughly tested. The results have been discussed concerning
urea concentration analysis in human blood.
Introduction. Enzyme biosensors can play the major role in the fields of growing
interest such as biomedicine and environmental science. These sensors seem to
be very promising for such a purpose, since analytical systems based on them
are simple, rapid and selective. The determination of urea in body fluids is one
of the most frequent analysis in routine clinical laboratory. In mammalian liver,
nitrogen derived from amino acid breakdown is converted to urea via a special
catabolic pathway, the urea cycle, and is readily excreted by the kidneys [1 ].
An increased concentration of urea in blood and its reduced level in urine is
therefore a strong indication for the renal disfunction.
In this work the alternative method for urease investigation based on a
thin-film conductometric microsensor is proposed. The application of micro-
fabricated conductometric transducers with thin-films interdigitated electrodes
for the enzyme biosensors has been presented recently [2, 3]. The main
advantage of conductometric detection is that almost all enzymatic reactions
involve either consumption or production of charged species and therefore lead
to the change in the ionic composition of the solution sample [4], From the
technological point of view it is important that thin-film metal electrodes are
suitable for miniaturization and large scale production using inexpensive
thin-film technology.
Although there is a great amount of information on the creation of
biosensors for urea determination, the prospects for their practical use are not
yet entirely clear. It is known that different factors of the analyzed liquid, blood
in particular, affect the sensor characteristics in many different ways, and this
effect should be taken into consideration when the analysis procedure is
developed [5]. Therefore, we found it expedient to investigate thoroughly the
properties of soluble and immobilized urease when they are influenced by
*Correspondence address.
© S. V. D Z Y A D E V I C H , G. A. Z H Y L Y A K , A- P. SOLDATKJN, A. V. EL 'SKAYA, 1996
S. V. D Z Y A D E V I C H ET AL.
Fig. 1. The calibration curves for the urea conductometric biosensors with soluble (a) and immobilized
(b) urease in the kinetic mode of measurement in 5 mM potassium phosphate buffer, p H 7.4
factors typical for biological liquids, namely, ionic strength, buffer capacity and
pH.
Materials and methods. Materials. Urease (EC 3.5.1.5, type B) with
activity 12 L.U./mg was obtained from Olina (Lithuania), bovine serum albumin
(BSA) was purchased from Sigma (USA) and 25 % aqueous solution of
glutaraldehyde (GA) was va (Germany). All other reagents were of analytical
grade.
Enzyme immobilization. Urease was immobilized using the procedure
described earlier [5, 61. 10 % (w/w) solutions of urease and BSA were
prepared in 5 mM phosphate buffer (KH 2P0 4 — NaOH), pH 7.4. Prior to the
deposition on the sensor chip these solutions were mixed in 5 % GOD, 5 %
BSA and glycerol was added till 10 %. Glycerol prevents a loss of the enzyme
activity during the immobilization and provides homogeneity of the membrane
and its better adhesion to the sensor surface. Since a differential experimental
set-up was used, a drop of the enzyme-containing mixture was deposited on the
sensitive area of the measuring pair of electrodes while only a mixture
containing 10 % BSA and 10 % glycerol was deposited on the reference pair
of electrodes. Then the sensor was placed in a saturated GA vapour for 30 min.
After exposure to GA, the membranes were dried at room temperature for 15
min. Before use the membranes were soaked in a 5 mM phosphate buffer, pH
7.4, for at least 30 min to equilibrate the membrane system.
Sensor design and measurements. The conductometric transducers consist
of two identical pairs of gold interdigitated electrodes photolithographically
patterned on a ceramic support with dimensions 5 mm * 30 mm (thickness 0.5
mm). The intermediate layer of titanium (0.1 /ит thick) is first deposited for
better gold adhesion. The sensitive area of each pair of electrodes forming a
conductometric transducer is about 1 mm * 1.5 mm.
The original device consisting of an «Emocon-2» apparatus (production of
Emocon Ltd, Kiev, Ukraine) coupled with the personal computer, a generator
and an amplifier has been developed for the automatic monitoring of the
biosensor system described. The generator provides the sinusoidal wave of
10.9 kHz frequency and 10 mV peak-to-peak amplitude about 0 V which is
applied to two pairs of electrodes forming a miniaturized conductance cell. The
low noise differential instrument — an amplifier measures the differential signal
between the pair of electrodes covered with the immobilized enzyme and those
covered with the «blank» membrane. The signal is monitored on IBM personal
computer.
The measurements were carried out in daylight at room temperature
(20—25°C) in a glass cell of about 2 ml volume. The interdigitated electrodes
were immersed in vigorously stirred buffer solution and after stabilization of the
54
C O N D U C T O M E T R I C UREASE MICROBIOSENSOR
The typical calibration curves for the urea biosensors (with soluble and
immobilized urease) in kinetic mode of measurement are shown in Fig. 1. These
curves were replotted in the Eadie-Hoffstee coordinates and revealed the kinetic
parameters enzymatic reaction. The hydrolysis kinetics for both immobilized
and soluble enzymes is described by the Michaelis-Menten equation. Km
appeared to be identical to that measured by widely used methods [4, 7, 8 ]
and its value was 1.33 mM for soluble urease and 3.73 mM for immobilized one
correspondingly. This result suggests that reaction in the presence of the
immobilized urease is controlled not only by the catalytic rate but also by the
diffusion of substrates and products through the membrane.
The dependence of the enzymatic reaction maximum rate (kinetic response)
on pH of the examined solution is presented in Fig. 2. In the case of the soluble
urease, a maximum was observed at pH 6.5 in 5 mM potassium phosphate
buffer. It does no with the results that we obtained earlier by the colorimetric
determination of urea in 100 mM potassium phosphate buffer where the
optimum pH for soluble urease was 7—7.5 [8 ]. This can be explained by noting
that at low buffer capacity of the ammonia ions can not associate completely
with the buffer species which are present in the sample. Thus, the enzyme
actually works at higher pH than that of examined solution and it is just the
reason of the apparent pH shift. In case of immobilized urease the optimum pH
is wider which can be explained by the enzyme stabilization in the membrane.
The dependence of the reaction rate on the buffer concentration was also
studied. In these experiments KC1 was added to the solutions of different buffer
concentration to ensure their identical conductivity. As seen from Fig. 3 the
kinetic response for soluble urease greatly decreased with an increase of the
buffer capacity of the solution. It means that the buffer species in the solution
can associate with ammonia ions. For immobilized urease kinetic response
Fig. 2. pH dependence of the relative value of the enzymatic reaction rate for soluble (curve / ) and
immobilized (curve 2) urease, 1 mM urea in 5 mM potassium phosphate buffer
Fig. 3. The dependence of the relative value of the enzymatic reaction rate for soluble (curve / ) and
immobilized (curve 2) urease on the potassium phosphate buffer concentration, pH 7.4. In each case
0.5 mM urea was added
sensor output signal, the portion of substrate was added to the cell.
Results and discussion. In the case of urea assay, the resulting conductivity
changes are caused by enzymatically catalyzed hydrolysis of the substrate:
55
remains practically constant within the whole range of the buffer concentration
tested.
Since biological liquids are featured by high concentrations of various salts
it seemed important to ascertain whether the response of the conductometric
urease biosensor depends on ionic strength. The dependence of the enzymatic
reaction rate on the KC1 concentration in the sample is shown in Fig. 4 for both
soluble and immobilized ureases. As for the effect of KC1 concentration on
kinetic response of urease biosensor it was detected that an increase of the salt
concentration within the 0 to 100 mM range resulted in about 80 % reduction
of the signal value. Further increasing of the salt concentration up to 400 mM
slightly changed the response value. From a practical point of view it is
important to note that for the salt concentration above 100 mM the kinetic
response of urease biosensor is practically independent on the ionic strength.
Conclusion. The enzymatically catalyzed hydrolysis of urea by the enzyme
immobilized on biosensor chip is also described by the classic laws of enzymatic
kinetics. The urease biosensor examined can be used for the determination of
the urea concentration in biological liquids. Since urea concentration in human
blood is much higher at pathologic states than in the normal (6—8 mM) [91.
For a blood sample diluted 20 times, the range of urea concentration falls within
the operating region of the created biosensor. The content of low-molecular
buffer components in human blood does not exceed 30 mM; therefore, after
diluting a blood sample by the buffer, the capacity of the obtained solution is
mainly determined by the diluting buffer. The ionic strength of the analyzed
solution required for the biosensor response stabilization may be ensured by
KC1 introduction into the solution until the final concentration 100 mM. Under
such conditions, the difference of the analyzed blood specimens in the ion
composition will not affect the value of the biosensor response to urea, and thus,
it will not distort the analytic results.
Acknowledgements. A part of this work was supported by a grant from
State Committee of Ukraine on Science and Technology (grant No. 5.3/21).
С. В. Дзядевич, Г. О. Жиляк, О. П. Солдаткін, Г. В. Єльська
Кондуктометричний уреазний мікробіосенсор для визначення сечовини на основі тонкошіівкових
гребінчастих електродів
Резюме
Вивчено характеристики кондуктометричного уреазного біосенсора для визначення сечовини на
основі тонкоплівкових гребінчастих електродів. Уреазу іммобілізовали ковалентною зшивкою з
сироватковим альбуміном бика за допомогою глутарового альдегіду. Результуючі зміни провід-
Fig. 4. The dependence of the relative value
of the enzymatic reaction rate for soluble
(curve 1) and immobilized (curve 2) urease
on the KC1 concentration. Measuring
conditions: 5 mM phosphate buffer, p H 7.4,
urea concentration — 1 mM
S V. D Z Y A D E V I C H ET AL.
56
CONDUCTOMETRIC UREASE MICROBIOSENSOR
UDC 577.15+573.6 Received 12. 05. 95
57
ності викликано ферментативним гідролізом сечовини. Згаданий процес як для іммобілізова
ного, так і для вільного ферменту описується класичними законами ферментативної кінетики.
Досліджено вплив на величину відгуку іонної сили, рН та буферної ємності розчину. Розглянуто
питання стосовно визначення концентрації сечовини у крові людини.
С. В. Дзядевич, Г. А. Жиляк, А. П. Солдаткин, А. В. Ельская
Кондуктометрический уреазный микробиосенсор для определения мочевины на основе
тонкопленочных гребенчатых электродов
Резюме
Изучены характеристики кондуктометрического уреазного микробиосенсора для определения
мочевины на основе тонкопленочных гребенчатых электродов. Уреазу иммобилизовали ковален-
тной сшивкой с бычьим сывороточным альбумином с помощью глутарового альдегида. Результи
рующее изменение проводимости вызвано ферментативным гидролизом мочевины. Этот про
цесс как для иммобилизованного, так и для свободного фермента описывается классическими
законами ферментативной кинетики. Исследовано влияние на величину отклика ионной силы,
рН и буферной емкости раствора Рассмотрены вопросы, связанные с определением концентра
ции мочевины в крови человека.
|