Development of Multi-parameter Analyser based on Electrochemical

Urea Biosensors and Electrolyte Electrodes for Monitoring of

Hemodialysis Patients

Julija Razumiene

, Vidute Gureviciene

, Marius Dagys

, Ieva Sakinyte

, Algimantas Jonuska

Laurynas Rimsevicius

, Svitlana Marchenko

and Alexey Soldatkin

Institute of Biochemistry, Vilnius University, Mokslininku 12, 08662, Vilnius, Lithuania

Nephrology Centre,

Vilnius University, Santariskiu 2, 08661 Vilnius, Lithuania

Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics, National Academy of Sciences of

Ukraine, Zabolotnogo Street 150,03143, Kyiv, Ukraine

Keywords: Biosensor, Amperometric Urea Detection, Potentiometric Urea Detection, Urease, Sodium and Potassium

Electrodes, Blood Dialysis.

Abstract: The idea of developing multi-parameter urea analyser comprising urea, Na

and K

selective electrodes has

been considered. For this purpose the urea biosensors based on urease and recombinant urease working in

amperometric and potentiometric way were developed. The working parameters of both urea biosensors were

studied and optimized. Possibilities of real samples analysis using the developed biosensors were shown by

measuring urea concentrations in blood dialysate taken from patients with renal failure. Both the

potentiometric and the amperometric biosensors demonstrated high degree of signal reproducibility (the

relative standard deviation of responses did not exceed 5 %). Change of sodium and potassium concentrations

during blood hemodialysis is dangerous life-threatening condition and their monitoring is an important feature

of point-of-care analyser. For this purpose high integrity commercial Na

and K

selective electrodes were

analysed and our own signal amplification and processing system proposed.

1 INTRODUCTION

Urea is the final product of protein metabolism and it

is synthesized in the liver as a result of amino acid

deamination (Kuralay et al., 2005). Excessive urea in

organism is excreted by renal system during blood

filtration, and elevated levels of urea concentration in

blood or serum usually indicate dangerous kidney

disease. Regular level of urea in serum varies from 15

to 40 mg/dl (2.5 – 6.7 mM), while in patients

suffering from renal failure urea concentrations in

serum can reach 180 – 480 mg/dl (30 – 80 mM), and

patients with such elevated concentrations have to

undergo blood dialysis treatment (Dhawan et al.,

2009). It is a dangerous condition – 5 year survival of

men older than 64 years who are starting dialysis is

worse than that of men with colon cancer and prostate

cancer (Parfrey and Foley, 1999).

For such reasons the methods for assessment of

urea concentration in blood, serum and spent

dialysate solutions are being developed. Urea

measurements are important in medical diagnostics

for clinical evaluation of renal function and

monitoring the effectiveness of dialysis treatment.

One of the first methods for urea determination was

based on spectrophotometric measurements after

sample treatment with specific compounds leading to

coloured solution with distinctive spectra (Patton and

Crouch, 1977; With et al., 1961). Such methods are

fairly accurate and are being used in medical practice,

but they are not suitable for real-time sample analysis.

Alternative technologies for urea measurements

being developed are based on biosensor electrodes.

They have different design approaches such as

potentiometric (Kuralay et al., 2005, Liu et al., 1993;

Ahuja et al., 2011; Boubriak et al., 1995),

conductometric (Soldatkin et al., 2014; Chen et al.,

1994; Sangodkar et al., 1996) and amperometric

(Sangodkar et al., 1996; Tiwari et al., 2009), they

usually employ urease (EC 3.5.1.5) as a catalyst for

urea breakdown, which is immobilized by a number

of methods on electrode surface (Dhawan et al.,

2009). The urease catalyses the hydrolysis of urea to

Razumiene, J., Gureviciene, V., Dagys, M., Sakinyte, I., Jonuska, A., Rimsevicius, L., Marchenko, S. and Soldatkin, A.

Development of Multi-parameter Analyser based on Electrochemical Urea Biosensors and Electrolyte Electrodes for Monitoring of Hemodialysis Patients.

DOI: 10.5220/0005814100930101

In Proceedings of the 9th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2016) - Volume 1: BIODEVICES, pages 93-101

ISBN: 978-989-758-170-0

yield ammonia and carbamic acid (equation 1) which

spontaneously decomposes into carbonic acid and a

second ammonia molecule (equation 2) (Carter et al.,

2009), as shown below:

N-C(O)-NH

O+H

→NH

N-COOH

(1)

N-COOH+H

O→NH

+HCO

(2)

The principle of enzyme based action of

amperometric and potentiometric detection systems

are presented in figure 1.

Monitoring of electrolyte composition in blood

during dialysis treatment is also very important.

Serum electrolytes (sodium, potassium, calcium,

phosphate) are usually elevated in chronic dialysis

patients, they have a high survival risk ratio (Iseki et

al., 1996). While sodium and potassium can be auto-

regulated during long dwell continuous ambulatory

peritoneal dialysis (CAPD) (Nolph et al., 1980),

patients undergoing hemodialysis exhibit high

incidence of cardiac arrhythmias during dialysis days

particularly due to sudden imbalance of blood

electrolytes (Ramirez et al., 1984). While the

strategies of retaining sodium and potassium levels in

blood and extracellular fluid are being constantly

developed (Much and Wilcox, 1982; Maduell et al.,

2013; Mc Causland et al., 2012), real-time monitoring

of sodium and potassium concentrations during

hemodialysis could be crucial to determine rapid

change of such electrolytes in order to take according

revival actions.

The purpose of the study is to find best sensor

candidates for point-of-care urea, sodium and

potassium analyser, useful for monitoring of patients

undergoing hemodialysis treatment. The electrodes

and bioelectrodes (biosensors) used in such

equipment should be accurate, not expensive, stable,

simple to use in field measurement, etc. In case of

possible urea biosensor, our scientific group until

recently has been developing several types of urea

biosensors (Boubriak et al., 1995; Soldatkin et al.,

2014; Mc Causland et al., 2012; Laurinavicius et al.,

2013). In this case a specific attention is paid to

previously developed and published potentiometric

(Kulys et al., 1986) and amperometric (Mc Causland

et al., 2012) urea biosensors that were further

developed, as described in this work. In case of

sodium and potassium measurements, we will adapt

to our needs one of many commercially available

electrolyte measurement system, consisting of

potentiometric flow-through electrode cell. It is used

in several electrolyte analysers, which electronics and

signal analysis algorithms are hardcoded into

microcontrollers, subject to copyright material, so in

this study we will devise our own signal amplification

and processing system.

2 EXPERIMENTAL

2.1 Chemicals and Reagents

In this work the enzyme used for assembly of urea

amperometric biosensors was urease from Canavalia

ensiformis (E.C. 3.5.1.5.), activity of 343.0 U/mg

from Calbiochem (Germany). The enzyme substrate

was used as the phosphate buffer solution, pH 7.2,

containing 1 M of urea. Thermally reduced graphene

oxide (TRGO) was used as electrode materials.

TRGO have been synthesized by us as proposed in

the protocol (Razumiene et al., 2015).

Figure 1: The principle of enzyme based action of amperometric and potentiometric urea detection systems.

Urease

BIODEVICES 2016 - 9th International Conference on Biomedical Electronics and Devices

For potentiometric biosensor creation

recombinant urease (R. urease, E.C. 3.5.1.5) from

USBiological (USA) expressed in E. coli, was used,

its activity was 150 U/mg. Bovine serum albumin

(BSA, fraction V) and urea were obtained from

Sigma-Aldrich Chemie; poly(vinyl alcohol)

containing styrylpyridinium (PVA-SbQ) from Toyo

Gosei Kogyo Co. Ltd (Japan). The working

phosphate buffer (KH

-NaOH), pH 7.4, was

prepared by using reagents from Helicon (Moscow,

Russia). Sensor chips with differential pair of pH-

sensitive field effect transistors produced at the JSC

“Kwazar” facilities (Kiev).

The samples of blood dialysate and serum for the

potentiometric measurements of urea content were

obtained from Kiev municipal scientific and practical

centre of nephrology and hemodialysis (Ukraine). All

others chemical reagents were obtained from Sigma-

Aldrich and were of analytical grade unless otherwise

mentioned.

2.2 Construction and Electrochemical

Measurements of Biosensors

2.2.1 Amperometric Urea Biosensors

Amperometric measurements were performed using

an electrochemical system PARSTAT 2273

(Princeton Applied Research) with a conventional

three-electrode system composed of an auxiliary

platinum plate electrode, a reference Ag/AgCl

electrode and working TRGO (Ø 3 mm) electrodes as

transducer for amperometric biosensor (Razumiene et

al., 2015). Aiming to design amperometric biosensor

TRGO were extruded by forming a tablet. The tablet

was sealed in a Teflon tube then tablet surface was

covering by the semipermeable terylene membrane

containing immobilized urease from Canavalia

ensiformi. The response of the prepared

amperometric biosensors to the addition of substrate

was investigated under potentiostatic conditions at

+200 mV (vs. Ag/AgCl) in a stirred phosphate buffer

solution, pH 7.2, 20

2.2.2 Potentiometric Urea Biosensor

Potentiometric biosensor was based on pH-sensitive

field effect transistors as current transducers

(Sheliakina et al., 2014; Pavluchenko et al., 2011).

Each transducer contained a differential pair of pH-

sensitive field effect transistors placed on a single

crystal with the total area of 8 mm × 8 mm. Signals

were recorded from both transistors and then signal

from reference transistor (covered with BSA

membrane) was subtracted from the signal of

transistor covered with enzyme membrane.

Transistors demonstrated pH-sensitivity of

approximately 40 mV/pH and transconductance of

400–500 mkA/V. More information about transistor

structure and their photo can be found in (Sheliakina,

2014) and description of a portative measuring device

– in (Pavluchenko et al., 2011).

A bioselective membrane on the transducer

surface was formed by immobilisation of R. urease in

PVA/SbQ photopolymeric membrane. 66 % of

PVA/SbQ and 10 % of R. urease were mixed at 1:1

ratio and the mixture (0,1 μl) was deposited on the

surface of the ISFET. Then sensor chip was exposed

under the UV lamp KF-4M (Ukrainian production) of

3.4V/m

for 20 min.

Measurements were carried out in the 5 mM

potassium phosphate buffer solution (KH

NaOH), pH 7.4, with intensive stirring at room

temperature. The biosensor and Ag/AgCl reference

electrode were placed into an open 1.5 ml measuring

cell. The urea concentrations in the working cell were

obtained by the addition of aliquots of concentrated

stock solution or real samples.

2.2.3 Na

and K

Selective Electrodes – Cell

Construction and Signal Processing

Measurements of electrolytes – sodium and

potassium ions – concentration in blood or standard

solutions were performed by using commercially

available potentiometric Sensa K and Sensa Na

electrodes in dedicated five electrode cell containing

integrated Sensa reference electrode, bubble detector

and three blank electrodes, forming a single flow-

through channel for measurement of solutions of

interest. The whole set was purchased from Sensa

Core Medical Instrumentation Pvt Ltd, India. The

idea of the study was to not use any dedicated

commercial electronic amplification and signal

analysis equipment, thus such system was designed

from ground up.

The voltamperometric measurements of

electrodes revealed that the resistance of sodium,

potassium and reference electrodes was in range of 4

– 10 MΩ, thus two stage, closed loop operational

amplifier (op-amp) based circuit was used to amplify

the signals – one op-amp was used to follow the

changes of electrode potential difference without

voltage gain, and the output signal was further

amplified at 11-fold gain by second op-amp. The gain

level was selected to allow optimal voltage

measurements with 18-bit analogue-to-digital

converter (ADC) (for example, MCP3424 from

Development of Multi-parameter Analyser based on Electrochemical Urea Biosensors and Electrolyte Electrodes for Monitoring of

Hemodialysis Patients

Microchip Technology Inc.), capable of measuring

potential differences up to 3.3 V in our electronic

setup. To avoid electromagnetic interference, op-amp

circuit was encased in dedicated small metal shield

box and placed on top of electrode contacts. The

constructed urea analyser employs ADCs with

dedicated computer hardware and software for signal

analysis and implementation of measurement

algorithms; however, in this study we used bench type

UT804 multimeter with USB data output (Uni-Trend

Group Limited, Hong Kong) for analysis of amplified

signals.

The measurements were performed by following

procedure. First, the electrode cell was assembled and

connected; inner fluid path and tubing were rinsed by

de-ionised water. Then the solution of interest was

aspired into fluid channel and amplified output signal

(voltage) was recorded by computer software

controlled multimeter. The fluid path was rinsed by

de-ionised water between measurements, after work

the electrodes were disassembled, their channels

dried and stored in room temperature. In case of

sodium ions concentration measurements, standard

referenc solutions with NaCl concentrations 100 –

200 mM were used to measure potential difference

dependence on electrolyte concentration. In case of

potassium ions concentration measurements, KCl

solutions from 2 to 7 mM were used, they also

contained 150 mM NaCl in order to maintain ionic

strength. In blood measurements, equal amounts of

solutions with different NaCl or KCl concentrations

or pure water were added to blood samples. In result

several batches of blood samples were obtained with

varying concentration of sodium and potassium by 10

or 1 mM, respectively.

3 RESULTS

3.1 Study of Amperometric Urea

Biosensor

Amperometric biosensor after addition of urea into

electrochemical cell shows substrate-dependent

anodic response. The biosensor response was fast:

90% of steady state current achieved in 20 s. The

response was measured as a difference between the

steady state and the background current. T

he urea

calibration curve with marked linear range is shown in

figure 2 and main characteristics of the biosensor are

shown in Table 1.

Figure 2: The urea calibration curve and the linear range

(solid line) obtained using the amperometric biosensor.

Applied electrode potential of 0.2 V, phosphate buffer

solution, pH 7.2.

Operational stability of the biosensors was tested by

consecutive measurements of the current response to urea

solution of 3 mM. At least twenty measurements of the

biosensor activity per day were done during three weeks.

Between the experiments the biosensor was stored at room

temperature. The averaged data are shown in figure 3.

Figure 3: Operational stability of the amperometric

biosensor.

As can be seen in figure 3 after a period of three

weeks the decreasing of sensitivity of the

amperometric biosensor was negligible.

The amperometric biosensor has been tested for

urea measurements in dialysate as well. Aiming to

validate responses of the biosensor measurements the

samples of dialysate in parallel were examined at the

hospital laboratory. The testing has been carried out

by investigating dialysate of four patients after one

hour of hemodialysis. Urea concentration data

obtained by both methods are presented in figure 4.

The results shown in figure 4 exhibited good

correlation between two methods.

BIODEVICES 2016 - 9th International Conference on Biomedical Electronics and Devices

Figure 4: Comparison of results of urea determination in

dialysate after one hour of hemodialysis. The dialysates of

four patients were examined by the amperometric biosensor

and in the hospital laboratory.

3.2 Study of Potentiometric Urea

Biosensor

Firstly an investigation of analytical characteristics of

the potentiometric biosensor based on R. urease at

determination of urea concentration in model

solutions was done. The calibration curve for urea

determination by the potentiometric biosensor is

presented in figure 5 and main characteristics of the

biosensor are shown in Table 1.

Figure 5: The urea calibration curve – dependence of

current versus final urea concentration. Inset shows

measurements in linear response region. Measurements

were performed in 5 mM potassium-phosphate buffer, pH

7.4, at room temperature.

The signal reproducibility of the biosensor was

studied, in order to verify electrode suitability for

measurement of urea concentration both in model

solution and real samples. For this task the biosensor

responses to urea concentration of 5 mM in model

solution and 150 µl of the serum or blood dialysate in

working buffer were recorded during one workday at

30 minutes intervals. As shown in Figure 6,

potentiometric biosensor demonstrated high degree

of signal reproducibility in all cases (the relative

standard deviation of responses did not exceed 5 %).

Figure 6: Reproducibility of responses of potentiometric

biosensor to model and real samples. Measurements were

performed in 5 mM phosphate buffer, pH 7.4, at room

temperature.

The developed biosensor based on R. urease was

tested by the analysis of samples of blood dialysate

taken from patients with renal failure. For this task the

concentration of urea was analyzed in 10 samples of

blood dialysate and in 10 samples of serum. The

samples were added to the working cells (10-fold

dilution), the responses of biosensor were measured

and compared with the previously plotted calibration

curve.

Figure 7: Correlation between the data on urea

concentration in real samples obtained by biosensor and

control method.

To check the accuracy of urea determination by

the biosensor, the samples were analyzed in a

diagnostic laboratory by the control method - the

commercial Vitros 250 (Johnson & Johnson)

analyzer, based on "dry chemistry" technology – urea

Development of Multi-parameter Analyser based on Electrochemical Urea Biosensors and Electrolyte Electrodes for Monitoring of

Hemodialysis Patients

content being measured by using urease reaction with

colorimetric detection of produced ammonia. The

results of biosensor analysis and those obtained by

control method are shown in figure 7. The study

revealed high correlation between methods used (R =

0.9917).

3.3 Characterization of Amperometric

and Potentiometric Urea

Biosensors

The most important characteristics in terms to apply

the amperometric or potentiometric biosensors in

analytical device are summarized in Table 1.

Table 1: Analytical characteristics of amperometric and

potentiometric urea biosensors.

Biosensor

characteristic

Amperometric Potentiometric

Linear range, mM 0.1-12 0.5-15

Operational range,

0.1-30 0.1-40

Detection limit, mM 0.1 0.1

Response

reproducibility

(relative standard

deviation of

responses), %

2-3 3-5

Response time, s 20 60-120

Sensitivity

after 20 days, %

100 110

Dilution of the

sample

10-20 10

References Present work

Marchenko et

al., 2015 and

present work

Comparison of characteristics of the

amperometric and the potentiometric biosensors

shows that both electrodes have adequate linearity

and operational range, detection limit and

reproducibility, which means they both could be

considered as good candidates for commercialization.

3.4 Studies of Na

and K

Selective

Electrodes

Commercial sodium and potassium electrodes were

used to analyse samples of standard solutions and

blood. This study was essential to mimic the

measurement procedure hardcoded into electrolyte

analysers which algorithms are sensitive copyright

material. However, the commercial electrodes we

purchased are being used in various applications,

each manufacturer having their own algorithms, and,

as consulted by representative of manufacturer, it is

legal to use them as long as one follows

recommended sample measurement procedure,

which is similar to ours as described before.

Consequently, the purpose of our study is to

analyse dependences of electrode potentials on

electrolyte concentrations in various solutions, also to

determine if our selected signal processing and

amplification method is adequate to obtain readable

data. Since the manufacturer provides calibration

information about each electrode and is responsible

for the quality of the product, we assumed that it is of

minor importance to perform extended time

dependent electrode response repeatability

measurements.

Figure 8: Sodium (top) and potassium (bottom) ion

selective electrode 11-fold amplified response dependence

on concentration. Standard solutions with known

concentrations are marked blue, blood with incremental

additions of sodium and potassium by 10 or 1 mM,

respectively, are marked red. Concentration of sodium or

potassium in unaltered blood samples are calculated from

measured potential and linear function from measurements

of standard solutions.

The measurements revealed that voltage signals

were stable (noise floor less than 2 mV of amplified

signal), immune to electromagnetic interference. In

BIODEVICES 2016 - 9th International Conference on Biomedical Electronics and Devices

fact, when one stage closed loop op-amp system with

direct 11-fold gain was used instead of two stage

system, signals were stable as well. After aspiration

of solution into electrode path, the signal in either

electrode setup asymptotically approaches stability in

2 – 5 minutes in exponential decay manner, at about

40 seconds the signal reaches 95 % of its final value.

For our study the responses were mathematically

fitted by exponential decay function, final offset

values were calculated and used in analysis.

Typical dependencies of amplified voltage

difference signals on electrolyte concentration from

measurements of blood and standard solutions are

presented in figure 8.

The study revealed that Na

and K

selective

electrodes responses exhibit linear concentration

dependence, as long as concentration range is within

the ones present in physiological media. The slope

values both in cases of standard and blood solutions

are similar. To estimate the best value, the device

algorithm should preferably contain exponential

decay minimization procedure; however,

measurement after waiting for about 40 seconds from

sample aspiration should be adequate.

3.5 Insights into Construction of

Multi-Parameter Urea Analyzer

Currently, urea measurement in blood and dialysate

samples are performed in laboratory using

commercial cumbersome equipment, the test itself is

termed and standardized as blood urea nitrogen

(BUN) measurement. Implementation of urea

biosensors (Dhawan et al., 2009) gives promising

leads to development of standalone point-of-care

compact urea measurement devices. Consequently,

our group together with colleagues has been

developing commercially viable biosensor designs

for about three decades (Boubriak et al., 1995;

Soldatkin et al., 2014; Maduell et al., 2013; Mc

Causland et al., 2012; Laurinavicius et al., 2013;

Kulys et al., 1986; Marchenko et al., 2015). This

study compares our best candidates to incorporate

into such device. Comparison of characteristics of the

amperometric and the potentiometric biosensors

shows that both electrodes have adequate linearity

and operational range, detection limit and

reproducibility, which means they both could be

considered as good candidates for commercialization.

However, several features of the amperometric sensor

such as short response time and higher accuracy due

to better linearity in relevant concentration range, as

well as technical benefits (lower sensitivity to

background noise, ease of production, almost

complete insensitivity to pH, etc.), not mentioned in

this study, make the amperometric urea sensor a

better option at this stage of designing of the multi-

parameter analyzer.

The study revealed that Na

and K

selective

electrode system is suitable for integration to device

as well. The signal amplification and analysis

techniques have been developed by technical cues

implied in sodium and potassium electrode cell

design. However, the amperometric biosensor and

ions selective electrode systems are of different

design nature and should be developed as different

parts of the multi-parameter analyzer. Specific care

must be taken when designing specimen and liquid

handling system, keeping in mind that electrical

amplification circuits would be separate – liquids

from both cells should not touch in order to avoid

signal interference from occurring common electric

plane.

4 CONCLUSIONS

The amperometric and potentiometric urea

biosensors based on urease and recombinant urease

were developed and possibilities of the real samples

analysis using the developed biosensors were shown.

The working parameters of the urea biosensors were

studied and optimized. Linear dynamic range of the

potentiometric urea determination was 0.5 – 15 mM,

detection limit - 0.1 mM. Urea concentrations were

determined in 20 samples of blood dialysate and

serum taken from patients with renal failure; the

potentiometric biosensor demonstrated a high

correlation of the results with the control method of

urea determination. Linear range of the amperometric

urea determination was 0.1 – 12 mM and detection

limit of 0.1 mM. The amperometric biosensor has

been tested for urea measurements in dialysate and

results correlated with data obtained in the hospital

laboratory. Both biosensors – the potentiometric and

the amperometric demonstrated high degree of signal

reproducibility in all cases (the relative standard

deviation of responses did not exceed 5 %). Thus,

both biosensors studied in this research can be

effectively used to diagnose the patients with renal

failure and to control the urea content in blood or

dialysis fluid during hemodialysis.

In this study the commercial sodium and

potassium ion selective electrodes were analysed

aiming to integrate them in to the designing analyser.

It was not used any dedicated commercial electronic

amplification and signal analysis equipment, thus,

such system was designed from ground up. The

Development of Multi-parameter Analyser based on Electrochemical Urea Biosensors and Electrolyte Electrodes for Monitoring of

Hemodialysis Patients

dependences of electrode potentials on electrolyte

concentrations in various solutions revealed that our

selected signal processing and amplification method

is adequate to obtain readable data.

Considering analytical and technical features of

the biosensor designs, it seems that benefits of the

amperometric sensor hold the edge over choosing the

latter in designing the commercial analyser. Together

with electrolyte electrodes, such multi-parameter

point-of-care blood and dialysis fluid analyser would

help in better outcomes and hemodialysis procedure

corrections for patients diagnosed with various stage

renal failures.

ACKNOWLEDGEMENTS

This work has been supported by Lithuanian Agency

for Science, Innovation and Technology Project E!

8835, National Academy of Sciences of Ukraine and

STCU project No. 6052.

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