Device for quantitatively determining urea content

Abstract

The group of inventions relates to the field of biotechnology and medicine, and more particularly to a coating for an electrode usable in analytical biosensing devices (biosensors) and to designs of such devices, and can be used for quantitatively determining urea content in body fluids, particularly saliva and urine. A coating for an electrode of a device for quantitatively determining urea content in body fluids comprises the following sequence of layers: a carbon nanomaterial; a polymeric material (3) based on a redox-active polymer; and a urease enzyme (4), wherein the redox-active polymer is electropolymerized polyazure, and the carbon nanomaterial is comprised of single-walled carbon nanotubes (2). A device for quantitatively determining urea content in body fluids comprises a graphite paste electrode (1), the surface of which is modified with the coating according to the claimed invention, said coating being secured by a dialysis membrane (5) with the aid of a plastic ring (6). The technical result consists in broadening the range of urea concentrations that can be detected in samples of body fluids (saliva and urine), improving the stability of the device for quantitatively determining urea content in body fluids by lowering to 7.2% the relative standard deviation of fifteen analytical signals received during consecutive measurements of the same concentration of urea, and increasing the long-term stability of the device to 15 days.

Description

[0001] DEVICE FOR QUANTITATIVE ASSESSMENT OF UREA CONTENT

[0002] Field of technology

[0003] The group of inventions relates to the field of biotechnology and medicine, namely to a coating for an electrode used in biosensor analytical devices (biosensors) and the designs of said devices, and can be used for the quantitative assessment of urea content in physiological fluids, in particular saliva and urine.

[0004] State of the art

[0005] In medical practice, the following colorimetric enzymatic analyzers are used to quantitatively assess the urea content in human blood serum (plasma) and urine: Urea-Olvex (Olvex Diagnosticum, Russia), Urea Color Abris+ (NPF ABRIS+, Russia), the Stat Fax 1904+ biochemical analyzer (Awareness Technology, USA), and the BioMajesty JCA-BM6070 / C automatic biochemical analyzer (JEOL Ltd, Japan). The disadvantages of these analyzers include high reagent consumption, in particular, the urease enzyme, the difficulty of performing quantitative analysis at home, and the impossibility of miniaturizing the analytical system.

[0006] More accessible for quantitative home testing are analyzers based on the principle of an amperometric enzyme biosensor. For example, the multifunctional "3 in 1 Meawsom" glucometer (Speedgluc, USA) uses test strips for uric acid, cholesterol, and glucose to quantitatively determine these biomarkers in human blood. Since urease is a hydrolase, the test strip must be modified to ensure the fastest possible detection of ammonia, which is formed during the enzymatic hydrolysis of urea.

[0007] A coating and a model of a device for quantitative assessment of urea content for medical diagnostic purposes are known, which contain a platinum electrode, the working surface of which is covered with a membrane of acrylonitrile-methyl methacrylate-sodium vinyl sulfonate copolymer, a layer of electrodeposited rhodium and a layer of urease immobilized in cross-linked bovine serum albumin [Velichkova Y., Ivanov Y., Marinov L., Ramesh R., Kamini N., Dimcheva N., Horozova E., Godjevargova T. Amperometric electrode for determination of urea using electrodeposited rhodium and immobilized urease / / Journal of Molecular Catalysis B: Enzymatic. 2011. V. 69. I. 3-4. PP. 168-175.]. The range of determined urea concentrations is 0.1 - 2.67 mM. The relative standard deviation of the analytical signal generated by the device during seven consecutive measurements of the same urea concentration (2 mM) is 5.1%.Long-term stability is 27 days, during which the biosensor response is 86.8% of the initial signal. The measurement time for one sample is 15 seconds.

[0008] Another example of a device for the rapid assessment of urea is a laboratory model that contains a graphite disk electrode, the working surface of which is modified with polyaniline and multi-walled nanotubes, the urease enzyme [Meibodi AS E., Haghjoo S. Amperometric urea biosensor based on covalently immobilized urease on an electrochemically polymerized film of polyaniline containing MWCNTs / / Synthetic Metals. 2014. V. 194. PP. 1-6]. The range of detectable urea concentrations is 0.07 - 10 mM. The relative standard deviation of the analytical signal generated by the device during five consecutive measurements of the same urea concentration (0.6 mM) is 2.6%. Long-term stability is 15 days, during which the biosensor response is at least 50% of the initial one. The measurement time of one sample is 50 seconds.

[0009] A significant drawback of these devices is the narrow range of detectable urea concentrations, which allows monitoring urea in the blood, where the concentration of this biomarker at rest is normally 2.5-9.2 mM [Adeyomoye O. I., Akintayo S. O., Omotuyi K. R., Adewumi AN The biological roles of urea: A review of preclinical studies / / Indian journal of nephrology. - 2022. - V. 32. - No. 6. - P. 539-545], while the concentration of urea in urine is much higher and normally is 150-500 mM \Adeyomoye O. I., Akintayo S. O., Omotuyi K. R., Adewumi AN The biological roles of urea: A review of preclinical studies / / Indian journal of nephrology. - 2022. - V. 32. - No. 6. - P. 539-545]. The closest analogue of the claimed group of inventions is a coating for the electrode of the device and a device for the quantitative assessment of the urea content in physiological fluids, known from the source [Lavrova TV. (Scientific supervisor - Kharkova A.S.).Formation of a biosensor based on urease immobilized in a bovine serum albumin-safranin-fullerene composite for the determination of urea / / XIX Regional Master's Scientific Conference (May 20-31, 2024): collection of papers]. The device contains a graphite-paste electrode, the working surface of which is covered with sequentially arranged layers of carbon nanomaterial in the form of fullerene, a redox-active polymer in the form of bovine serum albumin modified with safranin, and the urease enzyme, closed with a dialysis membrane and fixed to the electrode surface with a plastic ring. This device allows the determination of urea content in the concentration range from 68 to 410 mM. The relative standard deviation of fifteen analytical signals obtained in successive measurements of the same urea concentration is 10.5%.The long-term stability of the analytical system, i.e., the period during which the biosensor response is at least 50% of the initial value, is 4 days. The measurement time for one sample is 5 minutes. The main disadvantages of the device and coating described above are low long-term stability and a high standard deviation of the measurements.

[0010] Thus, the technical problem that the claimed group of inventions is aimed at solving is the insufficiently high technical characteristics of known coatings for the electrodes of devices and the design of devices for the quantitative assessment of the urea content in physiological fluids, in particular saliva and urine.

[0011] Disclosure of the essence of the invention

[0012] The technical result of the group of inventions is an expansion of the range of detectable concentrations of urea in samples of physiological fluids (saliva and urine), an increase in the stability of the device for the quantitative assessment of the urea content in physiological fluids by reducing the relative standard deviation of fifteen analytical signals obtained during successive measurements of the same urea concentration to 7.2%, as well as an increase in the long-term stability of the device to 15 days.

[0013] Long-term stability of the device should be understood as the period of time during which the biosensor response is at least 50% of the initial one.

[0014] The specified technical result is achieved in a coating for an electrode of a device for quantitatively assessing the urea content in physiological fluids, containing successively arranged layers of: carbon nanomaterial, polymer material based on a redox-active polymer and a urease enzyme, where electropolymerized polyazure is used as a redox-active polymer, and single-wall carbon nanotubes are used as a carbon nanomaterial.

[0015] In the particular case of implementation of the declared coverage:

[0016] - a layer of single-wall carbon nanotubes was obtained by applying a suspension of single-wall carbon nanotubes containing 1.0-2.0 wt.% of single-wall carbon nanotubes to the surface of the electrode;

[0017] - a layer of polymer material based on electropolymerized polyazure was obtained by electrochemical polymerization of a sodium-potassium phosphate buffer solution containing 0.2-0.3 wt.% azure I and 12.0-13.0 wt.% sodium sulfate (NaiSC) and subsequent drying;

[0018] - a layer of urease enzyme is obtained by applying a solution of urease enzyme containing 0.2-0.7 wt.% of urease enzyme to a layer of polymer material based on electropolymerized polyazure, and subsequent drying.

[0019] The said technical result is also achieved in a device for quantitatively assessing the urea content in physiological fluids due to the fact that it contains a graphite-paste electrode, the surface of which is modified with a coating in accordance with the claimed invention, secured to a dialysis membrane using a plastic ring.

[0020] One of the factors increasing the stability of enzyme systems in amperometric biosensors is the preservation of the catalytic activity of the enzyme and the stability of each component of the analytical systems. The increased stability of the claimed device is due to the use of a coating containing a layer of carbon nanomaterial in the form of single-wall carbon nanotubes instead of fullerenes, which are used in the closest analogue, since single-wall carbon nanotubes have higher mechanical strength than fullerenes, which increases the long-term stability of the entire analytical system [Devi N. et al. Carbon-based nanomaterials: carbon nanotube, fullerene, and carbon dots / / Nano-materials: Advances and Applications. - Singapore : Springer Nature Singapore, 2023. pp. 27-57].

[0021] The claimed electrode coating and device also comprise a polymeric material based on electropolymerized polyazure, while the coating in accordance with the closest analogue contains bovine serum albumin modified with safranin. When forming a polymeric material based on bovine serum albumin modified with safranin, glutaraldehyde is used as a cross-linking component, which promotes a change in the conformation of the urease enzyme and thereby reduces its catalytic activity, which reduces the long-term stability of the device [Diaz-Gonzalez J., Arriaga LG, Casanova-Moreno JR Probing the influence of crosslinkers on the properties, response, and degradation of enzymatic hydrogels for electrochemical glucose biosensing through fluorescence analysis / / RSC advances. 2024. V. 14. I. 14. PP. 9514-9528].

[0022] Brief description of the drawings

[0023] The group of inventions is illustrated by figures, where:

[0024] Figure 1 schematically shows the general appearance of the claimed device and the coating for the electrode of the device,

[0025] Figure 2 shows the registered signal from the claimed device in the form of a dependence of current strength (nA) on time (s),

[0026] Figure 3 shows the calibration dependence of the claimed device, Figure 4 shows the chemical structure of the claimed coating for the electrode.

[0027] The elements are designated on the figures by the following positions:

[0028] 1 - graphite-paste electrode,

[0029] 2 - a layer of single-walled carbon nanotubes,

[0030] 3 - a layer of polymer material based on electropolymerized polyazure,

[0031] 4 - urease enzyme layer, 5 - dialysis membrane,

[0032] 6 - plastic ring.

[0033] Implementation of the invention

[0034] The claimed coating for the electrode of a device for quantitatively assessing the urea content in physiological fluids contains the following sequentially arranged layers: a layer of carbon nanomaterial in the form of single-walled carbon nanotubes (2), a layer of polymer material (3) based on electropolymerized polyazure, and also a layer of the urease enzyme (4).

[0035] A layer of single-wall carbon nanotubes (2) is obtained by applying a suspension of single-wall carbon nanotubes, which contains predominantly 1.0-2.0 wt.% of single-wall carbon nanotubes, to the electrode surface. A layer of polymer material (3) based on electropolymerized polyazure is obtained by electrochemical polymerization of a sodium-potassium phosphate buffer solution, containing predominantly 0.2-0.3 wt.% of azure I and 12.0-13.0 wt.% of sodium sulfate (NaiSCU), and subsequent drying of the obtained layer. A layer of urease enzyme (4) is obtained by applying a solution of urease enzyme, containing predominantly 0.2-0.7 wt.% of urease enzyme, to the layer of polymer material (3), and subsequent drying of the obtained layer. The thickness of the above-described layers of the claimed coating for the electrode of the device can range from 0.5 to 1.0 mm.

[0036] The claimed device includes a graphite-paste electrode (1), the working surface of which is modified (coated) with the above-described coating, corresponding to the claimed invention, secured with a dialysis membrane (5) using a plastic ring (6).

[0037] Below are examples of the manufacture of the claimed device and the coating for the device electrode.

[0038] Example 1. To form a graphite-paste electrode (1), prepare a graphite paste containing the following components: graphite powder – 100 mg, mineral oil – 40 µl. The graphite paste is then added to the plastic body of the graphite-paste electrode (1), after which a platinum conductor is immersed in the graphite-paste-filled body. The surface of the electrode (1) is then coated with the claimed coating.

[0039] To obtain a coating in accordance with the claimed invention, the surface of the graphite paste of the graphite-paste electrode (1) is coated with a suspension of single-wall carbon nanotubes (2) in an amount of 10 μl, formed on the basis of 0.33 g of a dispersion of 1.0 wt.% single-wall carbon nanotubes and 500 μl of deionized water. A layer of polymer material (3) based on electropolymerized polyazure is then applied to the resulting layer of single-wall carbon nanotubes (2). To form the specified layer (3) by electrochemical polymerization, the graphite-paste electrode (1) is placed in a 30 ml measuring cuvette with a 0.05 M sodium-potassium phosphate buffer solution (pH = 5.6) containing 0.2 wt.% azure I and 12.0 wt.% sodium sulfate (NaiSCE) and connected to a potentiostat.Next, by cyclically changing the applied potential in the range from -0.2 to +1.3 V vs. the silver chloride reference electrode at a scan rate of 60 mV / s and stirring with a magnetic stirrer for 60 cycles, a uniform layer of polymer material (3) based on electropolymerized polyazure is formed. After which the graphite-paste electrode (1) is washed with a sodium-potassium phosphate buffer solution (pH = 6.8) and dried. A solution of urease enzyme (specific activity 200 U / mg) in an amount of 10 μl with a urease enzyme concentration of 0.2 wt.% is applied to the dried surface of the graphite-paste electrode (1) and left until completely dry. The resulting coating is fixed on the graphite-paste electrode (1) with a dialysis membrane (5) using a plastic ring (6).

[0040] Example 2. The formation of a graphite-paste electrode (1) is carried out similarly to Example 1. To obtain a coating in accordance with the claimed invention, the surface of the graphite paste of the graphite-paste electrode (1) is coated with a suspension of single-wall carbon nanotubes (2) in an amount of 10 μl, formed on the basis of 0.25 g of a dispersion of 1.5 wt.% single-wall carbon nanotubes and 16 μl of deionized water. Next, a layer of polymer material (3) based on electropolymerized polyazure is applied to the resulting layer of single-wall carbon nanotubes (2). To form the specified layer (3) by electrochemical polymerization, the graphite-paste electrode (1) is placed in a 30 ml measuring cuvette with a 0.05 M sodium-potassium phosphate buffer solution (pH = 5.6) containing 0.25 wt.% azure I and 12.5 wt.% sodium sulfate (Na2SO4) and connected to a potentiostat.Next, by cyclically changing the applied potential in the range from -0.2 to +1.3 V relative to the silver chloride reference electrode at a scan rate of 60 mV / s and stirring with a magnetic stirrer for 60 cycles, a uniform layer of polymer material (3) based on electropolymerized polyazure is formed. After which the graphite-paste electrode (1) is washed with a sodium-potassium phosphate buffer solution (pH = 6.8) and dried. A solution of urease enzyme (specific activity 200 U / mg) in an amount of 10 μl with a urease enzyme concentration of 0.5 wt.% is applied to the dried surface of the graphite-paste electrode (1) and left until completely dry. The resulting coating is fixed on the graphite-paste electrode (1) with a dialysis membrane (5) using a plastic ring (6).

[0041] Example 3. The formation of a graphite-paste electrode (1) is carried out similarly to Example 1. To obtain a coating in accordance with the claimed invention, the surface of the graphite paste of the graphite-paste electrode (1) is coated with a suspension of single-wall carbon nanotubes (2) in an amount of 10 μl, formed on the basis of 0.25 g of a dispersion of 2.0 wt.% single-wall carbon nanotubes and 6 μl of deionized water. Next, a layer of polymer material (3) based on electropolymerized polyazure is applied to the resulting layer of single-wall carbon nanotubes (2). To form the specified layer (3) by electrochemical polymerization, the graphite-paste electrode (1) is placed in a 30 ml measuring cuvette with a 0.05 M sodium-potassium phosphate buffer solution (pH = 5.6) containing 0.3 wt.% azure I and 13.0 wt.% sodium sulfate (NaiSCU) and connected to a potentiostat.Next, by cyclically changing the applied potential in the range from -0.2 to +1.3 V relative to the silver chloride reference electrode at a scan rate of 60 mV / s and stirring with a magnetic stirrer for 60 cycles, a uniform layer of polymer material (3) based on electropolymerized polyazure is formed. After which the graphite-paste electrode (1) is washed with a sodium-potassium phosphate buffer solution (pH = 6.8) and dried. A solution of urease enzyme (specific activity 200 U / mg) in an amount of 10 μl with a urease enzyme concentration of 0.7 wt.% is applied to the dried surface of the graphite-paste electrode (1) and left until completely dry. The resulting coating is fixed on the graphite-paste electrode (1) with a dialysis membrane (5) using a plastic ring (6).

[0042] The operating principle of the claimed group of inventions is as follows.

[0043] A potentiostat, a silver chloride reference electrode, a 5 ml measuring cuvette, a magnetic stirrer, and a laptop are additionally used for measurements. The silver chloride electrode and graphite-paste electrode (1) are immersed in the measuring cuvette so that the potassium-sodium phosphate buffer solution comes into contact with the immobilized biomaterial—the urease enzyme layer (4).

[0044] The device is connected via a platinum conductor to a potentiostat, which records the current versus time. A silver chloride electrode is connected to the potentiostat. Measurements are performed at a constant potential of +0.275 V and continuous stirring of the solution with a magnetic stirrer (300 rpm) at room temperature. After a stable current level is established, the required amount of the analyte solution (saliva or urine sample) is introduced into the measuring cuvette using a micropipette. After each measurement, the measuring cuvette is rinsed with potassium-sodium phosphate buffer solution (pH 6.8).

[0045] The urea concentration is calculated based on the current amplitude (sensor response, AI, μA) using a calibration curve previously constructed using a standard urea solution.

[0046] Examples of the results of calculating the concentration of urea in physiological fluids using the claimed group of inventions in accordance with the above examples 1-3 are given in Table 1.

[0047] Table 1. Results of calculating the concentration of urea in physiological fluids using the claimed group of inventions

[0048] The main characteristics of the claimed device for quantitative assessment of urea content and the device in accordance with the closest analogue are given in Table 2. Table 2. Comparative characteristics of devices for quantitative assessment of urea content in physiological fluids

[0049] Thus, the claimed coating for the electrode of the device and the device for the quantitative assessment of urea content in physiological fluids allow for the analysis of urea in the concentration range from 0.6 to 500 mM, while the time of one measurement is 4 minutes, the relative standard deviation of fifteen analytical signals obtained in successive measurements of the same urea concentration is 7.2%, and the long-term stability of the device is 15 days.

Claims

CLAUSES OF THE INVENTION 1. A coating for an electrode of a device for quantitatively assessing the urea content in physiological fluids, containing sequentially arranged layers of: carbon nanomaterial, polymer material (3) based on a redox-active polymer and a urease enzyme (4), characterized in that electropolymerized polyazure is used as the redox-active polymer, and single-wall carbon nanotubes (2) are used as the carbon nanomaterial.

2. A coating for the electrode of a device for quantitatively assessing the urea content in physiological fluids according to claim 1, characterized in that the layer of single-wall carbon nanotubes (2) is obtained by applying a suspension of single-wall carbon nanotubes to the surface of the electrode, containing 1.0-2.0 wt.% of single-wall carbon nanotubes.

3. A coating for an electrode of a device for quantitatively assessing the urea content in physiological fluids according to claim 1, characterized in that the layer of polymer material (3) based on electropolymerized polyazure is obtained by electrochemical polymerization of a sodium-potassium phosphate buffer solution containing 0.2-0.3 wt.% azure I and 12.0-13.0 wt.% sodium sulfate (NaiSCU) and subsequent drying.

4. A coating for an electrode of a device for quantitatively assessing the urea content in physiological fluids according to claim 1, characterized in that the layer of urease enzyme (4) is obtained by applying a solution of urease enzyme containing 0.2-0.7 wt.% of urease enzyme to a layer of polymer material (3) based on electropolymerized polyazure, and subsequent drying.

5. A device for quantitatively assessing the urea content in physiological fluids, comprising a graphite-paste electrode (1), the surface of which is modified with a coating according to any one of paragraphs 1-4, secured to a dialysis membrane (5) using a plastic ring (6).

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