WO2025116864A1

WO2025116864A1 - A reusable electrochemical sensor for urea determination in electrochemical applications and its preparation method

Info

Publication number: WO2025116864A1
Application number: PCT/TR2024/051406
Authority: WO
Inventors: Elif ESEN COSKUN; Munire Nalan DEMIR; Hılmıye Denız ERTUGRUL UYGUN
Original assignee: Dokuz Eylul Universitesi
Current assignee: Dokuz Eylul Universitesi
Priority date: 2023-11-27
Filing date: 2024-11-25
Publication date: 2025-06-05
Anticipated expiration: 2026-05-27

Abstract

The invention relates to an electrochemical sensor designed for instantaneous measurement of urea in a liquid sample. The sensor developed comprises a screen- printed electrode (SPE) (3) modified with gold nanoparticles (GNP), a cell (4) containing a phosphate buffer and KCI in which this SPE electrode (3) is immersed, and a potentiostat (2) that provides the connection between the SPE electrode (3) and the computer (1). The connection between this SPE electrode (3) and the potentiostat (2) is provided by the electrode connection part (8). The working electrode (7) of the SPE electrode (3) consists of two layers and contains gold nanoparticles in the first layer on the electrode surface and pyrrole-3-carboxylic acid (Py3CA), a molecularly imprinted polymer (MIP), coated on this first layer. Under this working electrode (7), there is a counter electrode (5) and a reference electrode (8), respectively. The sensor enables both precise and real-time urea determination by instantly reporting the resistance change caused by the complex formed when the Py3CA structure selectively reacts with urea. Electrodes washed with sulphuric acid after urea determination can be reused and thus a low-cost urea determination can be made.

Description

A REUSABLE ELECTROCHEMICAL SENSOR FOR UREA DETERMINATION IN ELECTROCHEMICAL APPLICATIONS AND ITS PREPARATION METHOD

Technical Field of the Invention

The invention relates to an electrochemical (impedimetric) sensor prepared for use in the measurement of urea in liquid samples using polymers prepared through molecular imprinting technology. In the invention, the rapid, sensitive and repeatable determination of urea, an important analyte for monitoring kidney functions, is made through an electrochemical sensor. The electrochemical sensor subject to the study can perform real-time measurements of urea molecules in liquid samples through molecularly imprinted polymers (MIPs). Thanks to the strong structures of the mentioned MIPs, urea determination can be repeated with high selectivity and without a decrease in measurement accuracy.

State of the Art

Urea, with the chemical formula CH4N2O, is an organic compound that can be naturally produced in the bodies of mammals, as well as being synthesized in laboratory environments. Urea, which is commonly used in fertilizers and is cheaper compared to other compounds, is utilized not only in the fertilizer industry but also in the pharmaceutical sector and animal feed production [1 ], Both artificially synthesized urea and urea naturally produced in the human body are of significant importance. Urea produced in the liver is formed through the conversion of toxic ammonia in the body into urea.

Ammonia, which is produced as a result of protein digestion primarily in the intestines and kidneys, is highly toxic to the body. Therefore, this ammonia is transported to the liver, where it is converted into urea through various reactions. Urea produced in the liver is then transported to the kidneys via the bloodstream and is excreted through urine. This process helps prevent the potential damage that ammonia could cause to the body. However, some genetic disorders or liver diseases can disrupt the production of urea. These disruptions make it more difficult or reduce the conversion of ammonia to urea. Such disruptions lead to the accumulation of ammonia in the body. Increased ammonia accumulation in the body can cause various serious diseases, particularly affecting the central nervous system [2], Another disruption in this cycle occurs when the produced urea cannot be excreted from the body, leading to an increase in the urea concentration in the blood. To prevent such serious conditions, urea measurements are performed during routine blood and urine tests.

Abnormal urea levels in blood and urine tests, such as high or low urea concentrations, can sometimes be caused by controllable factors like stress, poor nutrition, and dehydration. However, irregular urea values may also indicate a disruption in kidney function or serious conditions such as kidney failure [3] . In particular, a high urea level in the blood may signal that urea, produced in the liver and transported to the kidneys via the bloodstream, is not being filtered by the kidneys. Investigating why this filtration process, which is the primary function of the kidneys, is not being properly carried out is critical for the patient's health. Therefore, accurate and prompt urea measurements are of vital importance for early diagnosis and the implementation of necessary treatments.

Today, urea determinations are usually performed with routine blood tests. In these routine tests, the determination of urea in the blood is made under the name BUN, blood urea nitrogen. These tests are usually performed using spectrophotometric, enzymatic and kinetic methods. The results obtained are reported according to a specific reference range. These values give healthcare professionals an idea about the status of kidney functions. Based on the aforementioned urea values, decisions are made regarding whether further tests should be conducted, thereby enabling the early detection of any disorders. However, BUN (Blood Urea Nitrogen) measurements typically take about 3 hours for routine checks and 1 hour in emergency situations. Additionally, for the test to be performed, patients must have their blood drawn by healthcare workers before each test. This blood collection process can cause discomfort for patients.

In the patent application numbered US20140069823A1 in the state of the art, urea determination is made by enzymatic methods. One of the commonly used methods for urea determination, enzymatic methods, although effective in providing reliable results, also bring certain challenges. One of the primary issues is the difficulty in obtaining the necessary enzymes. These enzymes, which are acquired at high costs, require careful handling during transportation to the laboratory where the tests will be conducted to prevent the degradation of enzyme activity. Temperature control during transport is crucial to maintain the stability of these enzymes. This situation makes the transportation of enzymes more difficult and increases their already high prices even more. Another problem encountered during enzymatic tests is the sensitive experimental conditions that need to be prepared and protected. In order to ensure that the enzymes work in the correct activity, the pH values as well as the temperature value should be adjusted according to the enzyme used. During this pH adjustment, both time and material are lost and the possibility of human errors increases. The inability to obtain test results in less than a few hours also leads to further time loss. Additionally, the reuse rates of the mentioned enzymes are low. Therefore, the enzymes need to be procured repeatedly at regular intervals. Considering that these urea determinations are conducted daily on hundreds of thousands of patients as part of routine checks, and even multiple times a day on the same patient in some cases, tests performed using these enzymatic methods lead to a loss of both time and money.

The limitations and inadequacies of existing techniques have necessitated improvements in the methods used for urea determination. Factors such as the need to constantly maintain parameters like pH and temperature to prevent the stability of structures like enzymes used in urea determination from being affected, the low reuse rates of these enzymes requiring regular repurchase, the time loss caused by test results, the high costs of materials in the mentioned methods, and the discomfort experienced by patients during tests have all highlighted the need for advancements in these methods.

Brief Description and Aims of the Invention

The invention describes an electrochemical sensor that provides instantaneous electrochemical urea determination by means of MIPs (molecularly imprinted polymers) prepared in situ on electrodes and the preparation method of this sensor. Urea determination is achieved through the interaction between the molecularly imprinted polymer, Py3CA (pyrrole-3-carboxylic acid), located on the top surface of the electrochemical sensor prepared for this purpose, and urea. The Py3CA structure demonstrates selectivity towards urea and is reusable, enabling a low-cost and continuously operable method for urea determination. The Py3CA structure, which is the subject of the invention, performs the reaction with urea, shown below in Reaction 1.

Reaction 1

The aim of the invention is to provide an electrochemical sensor that has high selectivity against urea in liquid samples and performs real-time urea determination. In the parameters where chronoimpedance measurement is taken, instantaneous urea determination can be performed. In the invention, the Py3CA structure used in the electrochemical sensor is selective for urea, allowing the Py3CA on the electrode surface to bind exclusively to urea. The structure formed as a result of this binding increases the resistance. The change caused by the increase in resistance is detected by the sensor and the amount of urea in the liquid sample is determined based on this change. Electrochemical tests are performed using Palme Sense 3 potentiostat. PSTrace 5.7 interface software is used to make the measurements and calculate the impedance curves. Urea is detected with screen-printed electrodes (DRP110 Carbon electrode) with a three-electrode system (carbon working electrode, platinum counter electrode and Ag/AgCI reference electrode). Since this resistance change, which occurs due to the interaction between Py3CA and urea, can be detected instantly by the sensor, urea determination can be made in real time.

Another aim of the invention is to provide an electrochemical sensor that can perform repeatable urea determination. In the invention, the resistance resulting from the reaction between Py3CA and urea is used for urea determination and after the measurement, the electrode with the Py3CA-Urea complex on its surface is kept in sulphuric acid. As a result of this keeping process, urea moves away from the electrode surface and the Py3CA remaining on the electrode surface returns to its pre-reaction state. Since the Py3CA structure can easily return to its initial state after the measurement, urea determination can be repeated many times without losing its properties. In order to measure the repeatability of the printed sensor, 3 consecutive measurements were taken with the same electrode in 100 nM urea and it was observed that the sensor had good repeatability and the standard deviation was calculated as 100 ±1.37 nM.

Another aim of the invention is to provide an electrochemical sensor for sensitive urea determination. Sensitive urea determination can be made by means of the gold nanoparticles used in the coating of the first layer of the electrode surfaces used in the invention. By means of the inert structure of the gold nanoparticles, the particles do not react with the substances in the liquid samples and at the same time, by means of their high conductivity, they can transmit even the smallest resistance values formed in the presence of the Py3CA-Urea complex to the sensor. Thus, even if the Py3CA-Urea complex formed on the electrode surface is in a very low amount, it can be detected by the gold nanoparticles. Another aim of the invention is to provide an electrochemical sensor that can be used for selective, low-cost and rapid urea determination. By means of the MIP technology used in the invention, it is possible to selectively determine urea in complex biological matrices such as blood and urine without requiring any pretreatment. By means of the reusability of the Py3CA structure mentioned in the invention, urea determination can be made many times without the need to supply new materials, thus preventing the constant purchase of new materials. In addition, by means of the rapid formation of the complex between the Py3CA and urea, causing a resistance change, and the rapid transmission of this resistance change to the sensor by means of the high-conductivity gold nanoparticles, a rapid urea determination is carried out with the mentioned sensor.

Description of Drawings

Figure 1. Summary Form of Urea Determination

Figure 2. Au Modified SPE Form of Urea Determination

Figure 3. CV data of unmodified, printed and template removed electrodes; Red) bare GNPE-SPE, magenta) GNPE- Py3CA@Ur, blue) GNPE-Py3CA

Figure 4. SEM image of GNPE-Py3CA@Ur polymeric structure

Figure 5. XPS analysis scan spectra

Definition of Elements/Parts Composing the Invention

1. Computer

2. Potentiostat

3. Screen Printed Electrode (SPE)

4. Cell containing phosphate buffer and KCI

5. Counter electrode

6. Reference electrode

7. Working electrode 8. Electrode connection part

Detailed Description of the Invention

The invention relates to an electrochemical sensor for use in the determination of urea. The mentioned sensor comprises a screen-printed electrode (SPE) (3), a cell (4) containing a phosphate buffer and KCI in which the SPE electrode (3) is immersed, and a potentiostat (2) that provides the connection between the SPE electrode (3) and the computer (1 ). The connection between the SPE electrode (3) and the potentiostat (2) is provided by the electrode connection part (8). The working electrode (7) of the SPE electrode (3) consists of two layers and contains gold nanoparticles in the first layer on the electrode surface and pyrrole-3-carboxylic acid (Py3CA), a molecularly imprinted polymer (MIP), coated on this first layer. Under this working electrode (7), there is a counter electrode (5) and a reference electrode (8), respectively. By means of the high conductivity of the gold nanoparticles in the first layer, any resistance change occurring on the SPE electrode (3) is detected precisely and transmitted to the sensor instantly and quickly. B means of the high selectivity of the Py3CA structure in the second layer against urea, only Py3CA forms a complex with urea through the reaction specified in Reaction 1 , and after urea determination, it can return to its original form, making it possible to reuse the developed sensor.

Reaction 1 In the invention, Palm Sense 3 Potentiostat (2) is used to measure electrode surface properties, namely immobilisation stages and urea polymer binding and related concentration measurements with electrochemical impedance spectroscopy (EIS). When examining the parameters of the sensor, the measurements are obtained by immersing the electrode of the sensor into a cell (4) containing 50 mM PBS (phosphate buffer) at pH 7.0, Fe(CN)₆ ³7^4- redox probes, and KCI. With frequency scanning between 10000 Hz and 0.05 Hz, 180 mV is determined as a potential application of DC and AC, and the R1 (Q1 (R2W)) model is used as a circuit diagram to determine the impedance change in ohm values. The phosphate buffer (4) is used to keep the pH at the desired value. The redox probe is an organic or inorganic material that gives a reduction/oxidation reaction when it touches the electrode surface and is the chemical solution used to monitor the electrochemical properties of the electrode. KCI is used to increase the conductivity of the redox probe. Frequency and potential values are the values required for EIS measurement. The impedance of the counter electrode (5) consisting of a carbon with an area of 1 .6 cm² is one of the required measurement parameters. These values may vary depending on the electrode material and area size used. In the circuit diagram, R1 is the electrolyte resistance between the counter electrode (5) and the working electrode (7). R2 is the impedance value of the working electrode (7). Q is the capacitive load of the electrode surface. W is the mass transfer resistance of the electrolyte moving in the working electrode. The mentioned SPE electrode (3) is commercially procured as gold nanoparticle electrodes, which are prepared by depositing them onto the surface of a carbon electrode (5). By means of the high conductivity of these gold nanoparticles, any resistance change occurring on the electrode surface is rapidly transmitted to the sensor. Py3CA, a pyrrole-derived polymer with high selectivity against urea, is synthesised as a molecularly imprinted polymer on the surface of this gold nanoparticle-coated SPE electrode (3). In this synthesis process, Py3CA and template molecule urea structures are used as monomers in sulphuric acid medium and the synthesis is carried out in situ on the electrode surface. After the synthesis of this polymer, the sensor is ready for urea determination.

Preparation of the reusable, real-time electrochemical sensor used in the determination of urea, which is the subject of the invention, comprises the process steps of: i. in situ synthesis of the molecularly imprinted polymer Py3CA on the working electrode (7) on the gold nanoparticle-coated SPE electrode (3), and ii. removal of urea molecule from MIP material coated electrode. The electrochemical sensor in the invention enables rapid, sensitive, and realtime urea determination. The urea determination method using the electrochemical sensor in the invention, comprises the process steps of: i. dropping the liquid sample onto the working electrode (7) coated with gold nanoparticles and with Py3CA on the nanoparticle, which is on the SPE electrode (3), ii. forming Py3CA-Urea complex on the electrode, iii. transmitting the resistance change resulting from the formation of the Py3CA-Urea complex instantly to the sensor via gold nanoparticles, and iv. determining the amount of urea from the resistance change value using the potentiostat (2) and via the analyser/computer (1 ).

After each urea determination, the electrode is kept in sulphuric acid solution at a concentration of 100 mM for 30 minutes. During this keeping period, as stated in Reaction 2, urea is removed from the Py3CA-Urea complex and the Py3CA structure returns to the structure it had before urea determination. Covalent or non-covalent interactions may occur while forming molecularly imprinted structures. Non-covalent bonds are preferred more in terms of easy removal of the template molecule and reproducibility of the system. The acid used ensures the breaking of non-covalent bonds formed during the formation of the MIP material. Using the acid to remove the template molecule urea breaks non-covalent bonds by changing the pH value. In this way, urea determination can be repeated with the electrochemical sensor in the invention.

Reaction 2

Urea determination can be made without the need for a pre-treatment of the liquid sample (urine, blood or serum) in which the amount of urea is desired to be determined.

The urea measurement in this liquid sample is made through the resistance change caused by the complex formed as a result of the bonding between Py3CA, which is coated on the second layer of the electrode surface, and urea. While the carboxylic acid end (C=O-OH) on Py3CA interacts with the amine (NH2) ends on urea, the carbonyl (C=O) group on urea interacts with the imino (-NH) group on Py3CA and performs selectivity. The interacting ends polymerise around urea and the gaps formed become suitable for the three-dimensional structure of urea. Since this gap is suitable for the three-dimensional structure of urea, it can only interact with the urea molecule, like a key-lock fit. The formation of the Py3CA-Urea complex on the surface of the working electrode (7) causes a resistance change on the surface. This resistance change occurring in unit time is rapidly transmitted to the sensor by means of the gold nanoparticle with high conductivity located on the lower layer of the working electrode (7) and mentioned resistance change is measured by the sensor. In order to perform this measurement, the impedance value of the uncoated electrode is first taken. Then, after the electrode is coated and the urea is removed, the resistance change is followed by taking a separate measurement. As the immobilisation layer on the surface of the SPE electrode (3) increases, the redox probe reaching the surface decreases, causing the resistance to increase, and accordingly, as the concentration of the urea molecule in different samples increases, the sensor resistance will increase, and can be determined with the relevant invention. The change in the electrical charge distribution on the surface of the SPE electrode (3) is measured electrochemically and since these measurements follow the correlated changes in unit time, the developed measurement basis shows chrono impedimetric properties. At the same time, the working electrode (7) monitors the binding characteristics of urea to Py3CA on the surface of the working electrode (7) by electrochemical impedance spectroscopy (EIS). With this measurement, proteins and antigens in large masses that are not electroactive can be measured at very low detection ranges and very low detection limits. The detection limit and observable limit of urea in liquid samples are calculated with this sensor (n=3), using the equations LOD=3.3 (S)/m, LOQ=10 (S)/m, respectively, to calculate the LOD and LOQ values. According to these calculations, the LOD value is 3.9 nM and the LOQ value is 11.9 nM, and the regression coefficient is R²=0.9911. S in the equations is the standard deviation of the sensor response and m is the slope of the calibration curve.

The differences in anodic and cathodic potentials are examined with cyclic voltammetry (CV) measurement. When the anodic potential is examined, a decrease is observed, while an increase is observed in the cathodic potential. The reason for this difference is due to the decrease in conductivity on the surface. With the increase in the thickness of the material on the surface, the conductivity decreases and therefore a decrease in the current is observed. When the CV measurements are examined, it is seen that the electrode is modified and the decrease in the current observed after the desorption process proves that the template molecule is removed. The difference in the CV measurement made with bare-GNPE-SPE (Figure 3) due to the polymeric structure remaining on the electrode surface shows that the structure is not separated from the surface and can be reused.

In order to observe the coating of the electrode after electropolymerisation performed on the electrode surface, the SEM image of the GNPE-Py3CA@Ur polymeric structure was taken with Scanning Electron Microscopy (SEM) (Figure 4). The size of the structures formed after coating is approximately 55 nm.

With XPS analysis, the material surface is exposed to X-rays and photoelectrons are created by removing electrons from the atoms on the surface. The formed photoelectrons are measured in the electron analyser and the electron binding energies are determined. XPS analysis scanning spectra (Figure 5) are given, representing the peaks of C1s, N1 s and O1 s at ~285 eV, 400 eV and 532 eV for the electrode coated with GNPE-Py3CA@Ur and the GNPE-Py3CA electrode formed after the removal of urea. With this analysis, it is seen that the electrode preparation and desorption steps were successfully performed.

REFERENCES

[1] Finch, H.J.S.; Samuel A.M.; Lane, G.P.F.; Fertilisers anda Manures, Lockhart & Wiseman’s Crop Husbandry Including Grassland (Ninth Edition), Woodhead Publishing Series in Food Science, Technology and Nutrition, pg. 63-91.2014

[2] Sericin enhances ammonia detoxification by promotes urea cycle enzyme genes and activates hepatic autophagy in relation to CARD-9/MAPK pathway, HELIYON (2023), doi: https://doi.Org/10.1016/j.heliyon.2023.e21563.

[3] Dorum, S; Havali, C; Ure ddgusu bozukluklan klinik, laboratuvar ve genetik dzellikleri: Tek merkez deneyimi, MKU Tip Dergisi, 2022; 13(45)74-79

Claims

1. A real-time reusable electrochemical sensor for the determination of urea in liquid samples, comprising screen printed electrode (SPE) (3), a cell (4) containing phosphate buffer and KCI in which this SPE electrode (3) is immersed, the potentiostat (2) that provides the connection between the SPE electrode (3) and the computer (1 ), the electrode connection part (8) that provides the connection between the SPE electrode (3) and the potentiostat (2), the working electrode (7) consisting of two layers that the SPE electrode (3) has, and the counter electrode (5) and the reference electrode (8), respectively, under this working electrode (7).

2. An electrochemical sensor according to Claim 1 , characterized in that the mentioned liquid sample is blood, urine, or serum.

3. The preparation method of the electrochemical sensor according to claims 1 or 2, comprising the process steps of: i. in situ synthesis of the molecularly imprinted polymer Py3CA on the working electrode (7) on the gold nanoparticle-coated electrodes (3), and ii. removal of urea molecule from MIP material coated electrode.

4. An urea determination method performed using the electrochemical sensor according to claims 1 or 2, comprising the process steps of: i. dropping the liquid sample onto the working electrode (7) coated with gold nanoparticles and with Py3CA on the nanoparticle, which is on the electrode (3), ii. forming Py3CA-Urea complex on the electrode, iii. transmitting the resistance change resulting from the formation of the Py3CA-Urea complex instantly to the sensor via gold nanoparticles, and iv. determining the amount of urea from the resistance change value using the potentiostat (2) and via the analyser/computer (1 ).

5. A method according to Claim 4, wherein mentioned liquid sample is blood, urine or serum.