Electrode system for a device for quantitative analysis of blood glucose content

Abstract

The invention relates to the field of medicine, namely to biosensor analytical devices (biosensors) and can be used for continuous minimally invasive determination of blood glucose content. An electrode system for a device for quantitative analysis of blood glucose content comprises a reference electrode (6), an auxiliary electrode (5) and a working electrode (1) a surface of which is modified by a composite material containing a matrix (2) with chitosan covalently linked to an electron transport mediator, and inclusions of the glucose oxidase enzyme (3) placed in said matrix (2), wherein the matrix (2) of the composite material is chitosan-based, the surface of the working electrode (1) with the composite material is covered with a protective diffusion membrane (4), and the electrode system is designed for subcutaneous placement. The technical effect of the invention is to provide minimally invasive continuous determination of blood glucose concentrations within the range of 1.5 to 48 mM.

Description

[0001] ELECTRODE SYSTEM FOR A DEVICE FOR QUANTITATIVE ANALYSIS OF BLOOD GLUCOSE CONTENT

[0002] Pertinent art

[0003] The invention relates to the field of medicine, namely to biosensor analytical devices (biosensors) and can be used for continuous minimally invasive determination of glucose content in the blood.

[0004] Prior art

[0005] There is a known model of a device for quantitative assessment of glucose content in the blood, which is a screen-printed carbon electrode with an immobilized glucose oxidase enzyme [Khumngem, S., Jirakunakom, R., Thavarungkul, P., Kanatharana, P., & Numnuam, A. A highly sensitive flow injection amperometric glucose biosensor using a gold nanoparticles / polytyramine / Prussian blue modified screen-printed carbon electrode. Bioelectrochemistry. 2021, 138, 107718]. The device is based on the chemisorption of highly active glucose oxidase on gold nanoparticles adsorbed onto a layer of polythyramine. To fabricate the biosensor, a screen-printed carbon electrode modified with Prussian blue is coated. A distinctive feature of this device is its amperometric method for measuring blood glucose levels using the glucose oxidase enzyme on the printed electrode. The main drawback of this biosensor for quantifying glucose levels is the performance range of glucose concentrations from 0.001 to 1 mM, which only allows analysis by dilution.

[0006] The closest pertinent art of the claimed invention is the electrode system for a device for the quantitative determination of glucose levels in the blood, described in the Russian Federation Utility Model Patent RU228729 dated 09.09.2024. The device comprises an electrode system containing a reference electrode, an auxiliary electrode and a working electrode, the surface of which is modified with a composite material. The enzyme glucose oxidase is used as a biorecognition element. The composite material consists of tetraethoxysilane and contains chitosan covalently linked to the neutral red mediator. This device allows for the quantitative determination of glucose levels in the blood within the range of 0.01 to 0.92 mM, which also allows for analysis by dilution only.

[0007] Thus, the technical problem that the claimed invention aims to address is the low values within the range of glucose concentrations that can be determined using known electrode systems for devices of the specified purpose. Summary

[0008] The technical effect of the invention is providing minimally invasive continuous measurement of glucose concentrations in the blood within the range of 1.5 to 48 mM.

[0009] The specified technical effect is achieved in an electrode system for a device for quantitative analysis of glucose content in the blood due to the fact that it contains a reference electrode, an auxiliary electrode and a working electrode a surface of which is modified with a composite material containing a matrix with chitosan covalently linked to an electron transport mediator, and inclusions of the glucose oxidase enzyme placed in the specified matrix, wherein the matrix of the composite material is chitosan-based, the surface of the working electrode with the composite material is covered with a protective diffusion membrane, and the electrode system is designed for subcutaneous placement.

[0010] In a particular embodiment of the invention:

[0011] - neutral red or Nile blue is used as an electron transport mediator,

[0012] - the protective diffusion membrane contains a short-chain perfluoro sulfonic acid polymer, - the surfaces of the reference electrode and the auxiliary electrode of the electrode system are covered with a protective diffusion membrane.

[0013] The claimed electrode system, compared with the electrode system used in the device in accordance with the closest prior art, provides higher conductive properties due to the reduction of the area of the working electrode, since it is designed for subcutaneous placement, and thus ensures application in minimally invasive devices for continuous blood glucose monitoring. Phenazine Nile blue or neutral red mediators, used as part of the composite coating for the working electrode of the claimed electrode system, covalently cross-linked with chitosan, are effective electron transport mediators, allowing the conversion of the non-conducting polymer chitosan into a conductive redox-active polymer, and provide an increase in the sensitivity of the biosensor; and the said coating, made on the basis of a cross-linked chitosan polymer, is biocompatible and designed to immobilize the enzyme and mediator on the surface of the electrode.

[0014] Coating the electrodes included in the electrode system with a protective diffusion membrane serves to prevent the leaching of the components of the composite material from the surface of the working electrode into the subcutaneous layer, control the transport of the target analyte component into the enzyme membrane, and increase the selectivity for high-molecular-weight compounds.

[0015] Brief description of the drawings

[0016] The invention is explained by the figures, where:

[0017] Figure 1 schematically shows a rear view of the device for the quantitative analysis of blood glucose content with the claimed electrode system,

[0018] Figure 2 schematically shows a front view of the device for the quantitative analysis of blood glucose content with the claimed electrode system,

[0019] Figure 3 shows a sectional view of a part of the device for the quantitative analysis of blood glucose content with the claimed electrode system,

[0020] Figure 4 shows the recorded signal from the device incorporating the claimed electrode system as a function of current (nA) versus time (s),

[0021] Figure 5 shows the calibration curve of the device including the claimed electrode system, Figure 6 shows a linear section of the calibration curve.

[0022] The elements are indicated in the figures by the following positions:

[0023] 1 - working electrode of the electrode system,

[0024] 2 - matrix of composite material containing chitosan covalently linked to an electron transport mediator,

[0025] 3 - inclusions of the glucose oxidase enzyme,

[0026] 4 - protective diffusion membrane,

[0027] 5 - auxiliary electrode of the electrode system,

[0028] 6 - reference electrode of the electrode system.

[0029] Embodiment of the invention The claimed electrode system for a device for quantitative analysis of blood glucose content comprises a working electrode (1), a reference electrode (6) and an auxiliary electrode (5), and this electrode system is designed for subcutaneous placement. The electrode system can be applied on both sides to the substrate of the device for quantitative analysis of glucose content in the blood using a screen printing method. The working electrode (1) and the reference electrode (6) can be located on the front side of the device, and the auxiliary electrode (5) on the back side (Fig. 1, Fig.

[0030] 2).

[0031] The main materials of the specified electrodes (1, 5, 6) of the electrode system can be biocompatible materials containing electrically conductive particles of carbon, silver, gold or platinum.

[0032] The surface of the working electrode (1) of the claimed electrode system is modified (coated) with a composite material. The composite material contains a matrix (2) including a redox-active chitosan polymer covalently linked to an electron transport mediator, which can be neutral red or Nile blue, and inclusions of a biorecognition component represented by the glucose oxidase enzyme (3) placed in the matrix (2) (Fig. 3). The surface of the electrode system, including a reference electrode (6), an auxiliary electrode (5) and a working electrode (1) with the composite material applied to it is covered with a protective diffusion membrane (4). The protective diffusion membrane (4) can be made of perfluorosulfonic acid polymers, as well as silicone elastomers or polyurethanes.

[0033] An example of obtaining the claimed electrode system is given below.

[0034] A mixture consisting of 50 pl of a 1% chitosan solution in 1% acetic acid, 7.5 pl of a 25% aqueous solution of glutaraldehyde, 25 pl of a glucose oxidase enzyme solution (activity 50 U / mg, titer 0.2 g / ml) is prepared and mixed. Then a drop of 5 to 20 pl is formed on a glass slide and the resulting mixture is applied to the surface of the working electrode (1) by dipping it five times until a drop is formed, left until completely dry, then 2 pl of a protective diffusion membrane solution is applied on top and dried. The composition of the protective diffusion membrane solution can be a colloidal finely dispersed solution of a short-chain perfluorosulfonic acid polymer in a hydroalcoholic solution and a polymer content of 2 wt.%. After drying, the graphite microelectrode is immersed five times in the solution of the protective diffusion membrane so that the working (1), auxiliary (5) and reference electrodes (6) of the electrode system are covered. The operating principle of the claimed electrode system for a device for quantitative analysis of blood glucose content is as follows.

[0035] To register the biosensor response, one can use a micro potentiostat connected to an electrode system, the surface of the working electrode (1) of which is modified with a composite material containing a matrix (2) with chitosan covalently linked to an electron transport mediator, the enzyme glucose oxidase (3), placed in the said matrix (2) and covered with a protective diffusion membrane (4), as well as a portable computer or smartphone for connecting the micro potentiostat. The change in current (nA) versus time (s) as a blood sample or glucose-containing solution flows through the cell is taken as the microelectrode signal (see Fig. 4). Measurements are carried out at room temperature, with an operating potential of 0 V.

[0036] The glucose content in the sample is determined using a pre -built calibration curve shown in Fig.

[0037] 5. The calculation is carried out as follows: the glucose content in the cell is determined using an equation describing the linear section of the calibration curve shown in Fig. 6:

[0038] y=30x+530,

[0039] where x is the glucose concentration, mmol / dm3,

[0040] y is the biosensor response, nA.

[0041] Then the glucose content is determined in the sample. An example of calculations is given in Table 1.

[0042] Table 1. Results of calculating the concentration of glucose in the blood using a device for quantitative assessment of blood glucose content, including the claimed electrode system

[0043]

[0044] The main features of the device for quantitative assessment of blood glucose content, including the claimed electrode system, and the closest prior art device are shown in Table 2

[0045] Table 2. Comparative features of devices for quantitative assessment of blood glucose content

[0046]

[0047] Thus, the device for quantitative assessment of blood glucose content containing the claimed electrode system allows for continuous minimally invasive determination of blood glucose content with a concentration within the range of 1.5 to 48 mM while maintaining the duration of a single measurement of up to 2 minutes.

Claims

CLAIMS1. An electrode system for a device for quantitative analysis of blood glucose content, comprising a reference electrode (6), an auxiliary electrode (5) and a working electrode (1), a surface of which is modified with a composite material containing a matrix (2) with chitosan covalently linked to an electron transport mediator, and inclusions of enzyme glucose oxidase (3), placed in said matrix (2), characterized in that:the matrix (2) of the composite material is made of chitosan,the surface of the working electrode (1) with the composite material is covered with a protective diffusion membrane (4),and the electrode system is designed for subcutaneous placement.

2. The electrode system for a device for quantitative analysis of blood glucose content according to Claim 1, characterized in that neutral red or Nile blue is used as the electron transport mediator.

3. The electrode system for a device for quantitative analysis of blood glucose content according to Claim 1, characterized in that the protective diffusion membrane (4) contains a short-chain perfluorosulfonic acid polymer.

4. The electrode system for a device for quantitative analysis of blood glucose content according to Claim 1, characterized in that surfaces of the reference electrode (6) and the auxiliary electrode (5) of the electrode system are covered with a protective diffusion membrane (4).

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