Device for quantifying glucose concentration in bodily fluids

Abstract

The invention relates to the field of medicine, and more particularly to biosensor analytical devices and can be used for quantifying the concentration of glucose in blood, plasma and serum. The present device comprises a printed graphite electrode, the surface of which is modified with a hybrid composite material incorporating a biorecognition element in the form of glucose oxidase, wherein the hybrid composite material is a redox-active polymer based on chitosan covalently bonded to a mediator in the form of Nile blue. The technical result is that of broadening the range of detectable glucose concentrations to 0.006-1.0 мМ.

Description

[0001] DEVICE FOR QUANTITATIVE ASSESSMENT OF GLUCOSE CONTENT IN PHYSIOLOGICAL FLUID

[0002]

[0001] Technical field

[0003]

[0002] The invention relates to the field of medicine, namely to biosensor analytical devices and can be used for quantitative assessment of glucose content in blood, plasma and blood serum.

[0004]

[0003] State of the Art

[0005]

[0004] A device for quantitative assessment of glucose content is known, which is functionalized graphene on a screen-printed electrode, coated with gold sputtering and the enzyme glucose oxidase [Akhtar M. A. et al. Functionalized graphene oxide bridging between enzyme and Au-sputtered screen-printed interface for glucose detection / / ACS Applied Nano Materials. - 2019. - V. 2. - No. 3. - P. 1589-1596]. A characteristic feature of the device in question, common with the claimed invention, is an amperometric method for measuring glucose levels using the glucose oxidase enzyme on a printed electrode. A significant disadvantage of this device is its low sensitivity, which is 3.1732 pA-dm 3 / mmol'Sm 2 .

[0006]

[0005] The closest analogue of the claimed invention is a device for the quantitative assessment of glucose content in liquids, described in the Russian Federation patent for utility model RU218094, 11.05.2023. The device contains a graphite printed electrode modified with a hybrid composite material. The glucose oxidase enzyme is used as a biorecognition element. A conductive polymer based on thermally expanded graphite and an electropolymerized neutral red mediator is used as a hybrid composite material. The device allows for the quantitative assessment of glucose content in the range from 0.006 to 0.5 mM (mmol / dm 3 ) glucose, which in a number of cases is insufficient.

[0006] Thus, the technical problem that the claimed invention is aimed at solving is the insufficient sensitivity of devices for assessing glucose content.

[0007]

[0007] Disclosure of the essence of the invention

[0008]

[0008] The technical result of the solution is to expand the range of glucose concentrations determined to 0.006-1.0 mM.

[0009]

[0009] The stated technical result is achieved in a device for the quantitative assessment of glucose content in physiological fluids, due to the fact that it contains a graphite printed electrode, the surface of which is modified with a hybrid composite material containing inclusions of a biorecognition element in the form of glucose oxidase. Moreover, the hybrid composite material used is a redox-active polymer based on chitosan, covalently bound to a mediator in the form of Nile blue.

[0010]

[0010] In a particular case of the invention, a hybrid composite material is obtained by mixing a solution of chitosan in acetic acid, glucose oxidase, a solution of Nile blue and a solution of glutaraldehyde, applying the mixture to the surface of a graphite printed electrode and subsequent drying.

[0011]

[0011] A polymer base in the form of chitosan, used for immobilization of the enzyme glucose oxidase, creates a stable biocompatible environment that maintains the oxidative activity of the enzyme at a high level. The phenazine mediator Nile blue, covalently cross-linked with chitosan, is an effective electron carrier, allowing the transformation of a non-conducting matrix into a conductive redox-active polymer and providing increased sensitivity of the biosensor.

[0012]

[0012] Brief description of drawings

[0013]

[0013] The invention is illustrated by figures, where:

[0014]

[0014] Figure 1 schematically shows the claimed device,

[0015]

[0015] Figure 2 shows a sectional view of the device,

[0016]

[0016] Figure 3 shows the calibration dependence of the device,

[0017]

[0017] Figure 4 shows a linear section of the calibration curve of the device,

[0018]

[0018] Figure 5 shows typical device response patterns upon sample addition.

[0019] Implementation of the invention

[0019]

[0020] The claimed device comprises a graphite printed electrode (1), the working surface of which is modified (coated) with a hybrid composite material (2) containing inclusions of a biorecognition element (3) in the form of the enzyme glucose oxidase. The composite material (2) is a redox-active polymer based on chitosan, covalently linked (cross-linked) to a mediator, which is Nile Blue.

[0020]

[0021] To prepare the device, mix 10 µl of a 1% chitosan solution in 1% acetic acid, 25 µl of glucose oxidase enzyme, 5 µl of a saturated Nile blue solution, and 7.5 µl of a 25% aqueous glutaraldehyde solution in an Eppendorf tube and stir for 2 minutes. Apply 3 µl of the resulting mixture to the working surface of graphite printed electrode 1 and allow it to dry completely.

[0021]

[0022] The blood glucose device operates using the following principle. Measurements are performed using a potentiostat, a 5 ml measuring cell (cuvette), a magnetic stirrer, and a laptop computer. The device is placed in the cell, 5 ml of sodium-potassium phosphate buffer solution (pH 7.4) is added, the magnetic stirrer is turned on, and the background current in the measuring cell is recorded. To record the biosensor's response, it is preferable to use a CS150 potentiostat (Contest, China), connected to graphite printed electrodes and a personal computer. The physiological fluid sample to be analyzed is then injected. Measurements are performed at room temperature, at an operating potential of -500 mV, due to the oxidation-reduction properties of the hybrid composite material. Between measurements, the cuvette is rinsed with 5 ml of sodium-potassium phosphate buffer solution (pH 7.4). The signal of a graphite printed electrode is the dependence of current (nA) on time (s).The amplitude of the current change after introducing the sample into the measuring cuvette (biosensor response, AI, nA) is calculated. The calculation is performed as follows: the glucose content in the cell is determined using the equation describing the linear portion of the calibration curve (y = 1800x + 67.5, where x is the glucose concentration in the cell, mmol / dm3). 3 , y is the biosensor response, nA), then the glucose content is determined directly in the sample, taking into account dilution. An example of calculations is given in Table 1. After each measurement, the measuring cuvette is washed with 5 ml of phosphate buffer solution (pH 7.4).

[0023] Table 1

[0022]

[0024] Table 2 presents the characteristics of the closest analogue and the claimed device for quantitative assessment of blood glucose levels.

[0025] Table 2

[0023]

[0026] Thus, the claimed device for quantitatively assessing blood glucose levels increases the biosensor's sensitivity and determines glucose levels in diluted blood, plasma, or serum in the range of 0.006-1.0 mM. The glucose content in the sample can range from 0.5-45 mM, as the sample is diluted at least 50-fold during analysis. The low Michaelis constant of 0.9 mM indicates that the immobilized enzyme has a high affinity for glucose.

Claims

CLAUSES OF THE INVENTION 1. A device for quantitatively assessing the glucose content in physiological fluids, comprising a graphite printed electrode, the surface of which is modified with a hybrid composite material having inclusions of a biorecognition element in the form of glucose oxidase, characterized in that the hybrid composite material is a redox-active polymer based on chitosan, covalently bound to a mediator in the form of Nile blue.

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