Device for quantitative assessment of glucose content in physiological fluids

Abstract

The invention relates to the field of medicine, namely to biosensor analytical devices and can be used for quantitatively assessment of glucose content in blood, plasma and serum. The device comprises a graphite screen printed electrodes, whose surface is modified by a hybrid composite comprising an organosilicon matrix and inclusions of a biorecognition element in the form of glucose oxidase, as well as inclusions of carbon nanotubes and polyvinyl alcohol placed in the matrix. The organosilicon matrix consists of (3-aminopropyl)triethoxysilane and tetraethoxysilane in volume ratios of 20:80 and is covalently bound to a Nile blue mediator. The invention has the technical result of an increased upper limit of detectable glucose concentrations in physiological fluids up to 1.4 mmol / dm3.

Description

DEVICE FOR QUANTITATIVE ASSESSMENT OF GLUCOSE CONTENT INPHYSIOLOGICAL FLUIDSDESCRIPTIONTECHNICAL FIELD

[0001] The invention relates to the medicine, namely to biosensor analytical devices and can be used for quantitative assessment of glucose content in blood, plasma and serum.BRIEF DESCRIPTION OF THE PRIOR ART

[0002] There is a known device for a quantitative assessment of glucose content based on functionalized graphene assembled onto a gold- sputtered screen -printed electrode coated with the glucose oxidase enzyme [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, vol. 2, no. 3, pp. 1589-1596]. A characteristic feature of the device in question, which is common with the claimed invention, is an amperometric method for measuring glucose levels using the glucose oxidase enzyme on a screen-printed electrode. A significant disadvantage of this device is its low sensitivity, which is 3.1732 pA-dm3 / mmol-cm2.

[0003] [5] The closest prior art of the claimed invention is Patent RU218094 dated11.05.2023 disclosing a device for quantitative assessment of glucose content in fluids. The device comprises a graphite printed electrode modified with a hybrid composite. 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. The device provides quantitative assessment of glucose content in the range from 0.006 to 0.5 mM (mmol / dm3) of glucose, which is insufficient in some cases.

[0004] Thus, the claimed invention is aimed at solving the technical problem of insufficient sensitivity of devices for assessment of glucose content.SUMMARY OF THE INVENTION

[0005] The technical effect of the invention is an increase in the upper limit of the detectable concentrations of glucose in physiological fluids to 1.4 mmol / dm3.

[0006] The said technical effect is achieved in a device for quantitative assessment of glucose content in physiological fluids due to the fact that it comprises a graphite printedelectrode, whose surface is modified with a hybrid composite having inclusions of a biorecognition element in the form of glucose oxidase. The hybrid composite comprises an organosilicon matrix comprising (3-aminopropyl)triethoxysilane and tetraethoxysilane in the volume ratio of 20:80 and covalently bound to a mediator in the form of a Nile blue, as well as inclusions of polyvinyl alcohol and carbon nanotubes placed in the matrix.

[0007] In a particular embodiment of the invention, the hybrid composite is prepared by mixing (3-aminopropyl)triethoxysilane, tetraethoxysilane, glucose oxidase, NaF catalyst, polyvinyl alcohol solution and carbon nanotube suspension, applying the resulting mixture, a saturated solution of Nile blue and an aqueous solution of glutaraldehyde to the surface of a graphite printed electrode, and subsequent drying.

[0008] The organosilicon base (matrix) consisting of (3 -aminopropyl)triethoxy silane and tetraethoxysilaneis used for the glucose oxidase enzyme immobilization , and creates a stable biocompatible environment that maintains the oxidative activity of the enzyme at a high level, and further prevents leaching thereof from the electrode surface, which allows extending the operating time of the biosensor without replacing the sensitive element. The phenazine mediator (Nile blue)is covalently cross-linked with the matrix by the amino group of (3-aminopropyl)triethoxysilane, as an effective electron carrier, allows transforming the non-conductive organosilicon matrix into a conductive polymer. Inclusions of polyvinyl alcohol act as a structure-controlling agent. Multi-walled carbon nanotubes, due to their large surface area, significantly increase the electrical conductivity of the organosilicon matrix and serve as a "conductive bridge" between the enzyme molecules and the Nile blue mediator in possible places of spatial remoteness of the molecules from each other.Brief description of drawings

[0009] The invention is explained by the figures, where:Figure 1 schematically shows the claimed device,Figure 2 shows a section of the device,Figure 3 shows the recorded signal from the device as current strength (nA) vs. time (s),Figure 4 shows the device calibration curve, andFigure 5 shows a linear section of the calibration curve.

[0014] The elements are designated in the figures by the following reference numbers:1 - graphite printed electrode,2 - hybrid composite,3 - matrix of the composite,4 - inclusions of biorecognition element,5 - inclusions of carbon nanotubes,6 - inclusions of polyvinyl alcohol.DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

[0010] The claimed device comprises a graphite screen-printed electrode (1), whose working surface is modified (coated) with a hybrid composite (2). The hybrid composite (2) comprises an organosilicon matrix (3) and inclusions of a biorecognition element (4) in the form of a glucose oxidase enzyme, inclusions of multi-walled carbon nanotubes (5), and inclusions of polyvinyl alcohol (6) placed in the matrix.

[0011] The organosilicon matrix (3) is a polymer consisting of (3 -aminopropyl) triethoxysilane and tetraethoxysilane in volume ratio of 20:80, which is covalently crosslinked with a Nile blue mediator. Cross-linking of the polymer with the mediator occurs via the amino group of (3 -aminopropyl) triethoxy silane.

[0012] Polyvinyl alcohol inclusions (6) act as a structure-controlling agent due to the fact that polyvinyl alcohol is bound to the organosilicon matrix by hydrogen bonds.

[0013] To obtain the claimed device, 20 pL of (3-aminopropyl)triethoxysilane, 80 pL of tetraethoxysilane, 50 pL of glucose oxidase enzyme (titer 25 mg / ml, activity 100 E / mg), 5 pL of NaF catalyst (5% aqueous solution), 20 pL of 20% polyvinyl alcohol solution, 10 pL of 1% suspension of multi-walled carbon nanotubes are mixed in an Eppendorf test tube and stirred for 5 minutes. 3 pL of the resulting mixture, 2.4 pL of a saturated solution of Nile blue, 0.5 pL of a 25% aqueous solution of glutaraldehyde are applied to the working surface of the graphite printed electrode (1) and left until completely dry.

[0014] The operating principle of the device for assessment of glucose content in physiological fluids is as follows.

[0015] A potentiostat, a 5 mL cell, a magnetic stirrer and a portable computer are used for measurements. The device is placed in the cell, 5 mL of sodium-potassium phosphate buffer solution (pH 7.4) are added thereto, the magnetic stirrer is turned on and the background current in the measuring cell is recorded. The biosensor response can be recorded with a CS150 potentiostat (Contest, China) connected to graphite printed electrodes and a personal computer. Then the analyzed sample of physiological fluid is introduced. The measurements are carried out at room temperature and an operating potential of -500 mV, which is due to the redox properties of the hybrid material. Between measurements, the cuvette is washed with 5 mL of sodium-potassium phosphate buffer solution (pH 7.4). The signal of the graphite printed electrode (1) is the cunent strength (nA) vs. time (s) (see Fig. 3).

[0016] Next, the amplitude of the change in cunent strength after introducing the sample into the measuring cuvette is calculated (biosensor response, Al, nA). The glucose content in the sample is determined using the pre-built calibration curve shown in Fig. 4. The glucose content in the cell is calculated using the equation describing the linear section of the calibration curve shown in Fig. 5:

[0017] y = 115-x+23,

[0018] where x is the concentration of glucose in the cell, mmol / dm3,

[0019] y is the biosensor response, nA.

[0020] Then the glucose content is determined directly in the sample, taking into consideration the dilution factor. . An example of calculations is given in Table 1.

[0021] Table 1. Results of calculating the concentration of glucose in the blood using a device for quantitative assessment of glucose content in physiological fluids.

[0022] After each measurement, the measuring cuvette is washed with 5 mL of phosphate buffer solution (pH 7.4). The main features of the claimed device for assessment of glucose content in physiological fluids and the closest prior art device are shown in Table 2.

[0023] Table 2. Comparative features of devices for quantitative assessment of glucose content in physiological fluids

[0024] Thus, the claimed device for quantitative assessment of glucose content in physiological fluids allows extending the range of detectable glucose concentrations in diluted blood, plasma or serum to 1.4 mM.

Claims

CLAIMS1. A device for quantitatively assessment of glucose content in physiological fluids, comprising a graphite printed electrode, whose surface is modified with a hybrid composite having inclusions of a biorecognition element in the form of glucose oxidase, characterized in that the hybrid composite contains an organosilicon matrix comprising (3- aminopropyl)triethoxysilane and tetraethoxysilane in volume ratio of 20:80, and covalently bound to a Nile blue mediator, as well as inclusions of polyvinyl alcohol and carbon nanotubes placed in the matrix.

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