Material and device for assessing lactate content in body fluids

Abstract

A composite material contains: bovine serum albumin modified with neutral red; single-walled carbon nanotube inclusions; and lactate oxidase enzyme inclusions. A device for quantitatively assessing lactate content in body fluids comprises a working electrode, the surface of which is coated with the composite material according to the claimed invention. The technical result is that of increasing the upper limit of the range of detectable concentrations of lactate in body fluids to 57 mM, as well as reducing the time taken to perform a single measurement using the claimed device.

Description

[0001] MATERIAL AND DEVICE FOR ASSESSING LACTATE CONTENT IN PHYSIOLOGICAL FLUIDS

[0002] Field of technology

[0003] The group of inventions relates to the field of biotechnology and medicine, namely to composite materials for biosensor analytical devices (biosensors) and the designs of said devices, and can be used for the quantitative assessment of lactate content in physiological fluids such as blood and sweat.

[0004] State of the art

[0005] In medical practice, the following equipment is used to determine lactate: a device based on the principle of enzymatic amperometric measurement, trade mark LabTrend BST Bio Sensor (BST Bio Sensor Technologie GmbH, Germany), portable devices, for example, trade marks Lactate Scout (EKF - Diagnostic GmbH, Germany) and StatStrip Lactate (NOVA Biomedical, USA). Also used are more sensitive systems that require a smaller volume of blood sample for analysis, implemented in the following devices [Dagar K., Narwal V., Pundir C. S. An enhanced L-lactate biosensor based on a nanohybrid of chitosan, iron-nanoparticles and carboxylated multiwalled carbon nanotubes / / Sensors International. - 2023. - T. 4. - C. J 00245], [Thongkhao P. et al. Disposable Polyaniline / m-Phenylenediamine-Based Electrochemical Lactate Biosensor for Early Sepsis Diagnosis / / Polymers. - 2024. - T. 16. - No. 4. - P. 473].

[0006] All of the above devices are designed for invasive lactate determination in human plasma, serum, or blood. However, noninvasive sampling of bodily fluids, such as sweat, is more comfortable for the user.

[0007] The following model of a device for non-invasive diagnostics of lactate in human saliva is known, which contains a printed electrode, the working surface area of ​​​​which is covered with a suspension of zinc oxide and graphene oxide nanoparticles and the enzyme lactate oxidase [Han J., Shaohui J. Fabrication of a novel sensor for lactate screening in saliva samples before and after exercise in athletes / / Alexandria Engineering Journal. - 2024. - V. 92. - P. 171-175}. The range of detectable lactate concentrations is 0.015-1.25 mM. The relative standard deviation of the analytical signal generated by the device during seven consecutive measurements of the same lactate concentration (0.25 mM) is 2.9%.

[0008] Another example of a device for the non-invasive rapid assessment of lactate in human sweat is a laboratory model containing a printed electrode whose working surface area is coated with Prussian blue, taken as a redos-active compound, the lactate oxidase enzyme immobilized in the chitosan polymer [Plekhanova Yu. V., Reshetilov A. N. Biosensor for the simultaneous determination of glucose and lactate in human sweat / / Bulletin of Tula State University. Natural Sciences. - 2018. 11. 3. pp. 70-79]. The device demonstrated high efficiency of generating an analytical signal upon introducing lactate into the system and restoring the activity of the receptor element: the time of a single measurement of one sample is no more than 5 minutes. The linear range of the determined concentrations was 0.04 - 0.16 mM.

[0009] A significant drawback of these non-invasive diagnostic devices is that their sensitivity only allows for monitoring low lactate concentrations. This range of detectable lactate values ​​is suitable for assessing blood lactate levels, which normally range from 0.5 to 2.2 mM at rest [Crapnell, RD; Tridente, A.; Banks, C.E.; Dempsey-Hibbert, NC Evaluating the Possibility of Translating Technological Advances in Non-Invasive Continuous Lactate Monitoring into Critical Care. Sensors 2021, 21, 879], while the lactate concentration in sweat is significantly higher and normally ranges from 10 to 25 mM [Garcia-Morales R. et al. Bioengineered Lactate Oxidase Mutants for Enhanced Electrochemical Performance at Acidic pH / / ChemE-lectroChem. - 2023. - T. 10. -№. 22. - C. e202300296], and during intense physical activity it can reach up to 100 mM [Daboss E. V. et al.On-body hypoxia monitor based on lactate biosensors with a tunable concentration range / / Journal of Electroanalytical Chemistry. - 2023. - T. 935. - C. 117330].

[0010] The closest analogue of the claimed group of inventions is a composite material and a device for the quantitative assessment of lactate content in physiological fluids, described in [Ma G. Electrochemical sensing monitoring of blood lactic acid levels in sweat during exhaustive exercise / / International Journal of Electrochemical Science. - 2023. - Vol. 18. - No. 4. - P. 100064]. The said device contains a graphite printed electrode, the surface of which is coated with a nanomaterial in the form of graphene oxide, the lactate oxidase enzyme, which is immobilized in the polymer polyvinyl butyral, and a solution of potassium hexacyanoferrate (III) is additionally introduced into the system as a redox-active compound. The disadvantages of this device and composite material are that it only allows the determination of lactate at concentrations of up to 32 mM.The relative standard deviation of eight analytical signals obtained from successive measurements of the same lactate concentration was 3.50%.

[0011] Thus, the technical problem that the claimed group of inventions is aimed at solving is the insufficiently high technical characteristics of known composite materials and devices for the quantitative assessment of lactate content in physiological fluids, in particular sweat and blood.

[0012] Disclosure of the essence of the invention

[0013] The technical result of the group of inventions is an increase in the upper limit of the range of detectable lactate concentrations in physiological fluids to 57 mM, as well as a reduction in the time spent on performing a single measurement by the claimed device.

[0014] The specified technical result is achieved in a composite material for a device for quantitatively assessing the lactate content in physiological fluids, containing a polymer matrix, inclusions of a nanomaterial and inclusions of the lactate oxidase enzyme, immobilized in the specified matrix, wherein the polymer matrix contains a polymer modified with a redox-active compound, wherein bovine serum albumin is used as the polymer, neutral red is used as the redox-active compound, covalently bound to the specified polymer, and the nanomaterial is single-walled carbon nanotubes, wherein the composite material contains components in the following ratio, wt.%: inclusions of single-walled carbon nanotubes - 1.0-2.0, inclusions of the lactate oxidase enzyme - 2.0-3.0, the polymer matrix is ​​the rest.

[0015] In the particular case of implementation of the claimed composite material:

[0016] - the composite material was obtained by mixing bovine serum albumin, potassium-sodium phosphate buffer solution, suspension of single-walled carbon nanotubes, saturated solution of neutral red, lactate oxidase enzyme and glutaraldehyde solution.

[0017] The said technical result is also achieved in a device for quantitatively assessing the lactate content in physiological fluids due to the fact that it contains a working electrode, the surface of which is covered with a composite material in accordance with the claimed invention.

[0018] In a particular case of the implementation of the declared device:

[0019] - a graphite printed electrode or an inert metal electrode is used as the working electrode.

[0020] The dependence of the analytical signal of lactate oxidase-based biosensors on lactate concentration is typical for enzymatic reactions. Therefore, the experimental data are approximated by a two-parameter hyperbola equation and interpreted within the framework of the classical Michaelis-Menten kinetic model. The upper limit of detectable enzyme biosensor concentrations is determined by a parameter similar to the Michaelis constant. The components used to form the proposed device alter the microenvironment of the lactate oxidase enzyme, which alters the enzyme's affinity for lactate and leads to a higher substrate concentration required to achieve the same catalytic efficiency as the free enzyme.Thus, the increase in the upper limit of the range of detectable lactate concentrations by the claimed device compared to the device of the closest analogue is due to the fact that the claimed device uses a polymer matrix containing a polymer modified with a redox-active compound, where the polymer used is bovine serum albumin modified with neutral red, and inclusions of nanomaterial in the form of single-walled carbon nanotubes.

[0021] The use of single-wall carbon nanotubes as a nanomaterial results in a more developed working electrode surface. The immobilization of a redox-active compound in the form of neutral red directly within the polymer structure used to immobilize the lactate oxidase enzyme enhances the enzyme-redox-active compound interaction, leading to structural changes (steric hindrance) that affect the accessibility of lactate to the enzyme's active site and contribute to an increase in the upper limit of detectable lactate concentrations (Michaelis constant).

[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 composite material and device,

[0025] Figure 2 shows a general view of the claimed composite material and device in section,

[0026] Figure 3 shows the registered signal from the claimed device in the form of a dependence of current strength (nA) on time (s),

[0027] Figure 4 shows the calibration dependence of the claimed device,

[0028] Figure 5 shows the linear section of the calibration curve.

[0029] The elements are designated on the figures by the following positions:

[0030] 1 - working electrode,

[0031] 2 - inclusions of single-wall carbon nanotubes,

[0032] 3 - inclusion of the enzyme lactate oxidase,

[0033] 4 - polymer matrix.

[0034] Implementation of the invention

[0035] The claimed composite material contains:

[0036] - a polymer matrix (4) comprising a polymer in the form of bovine serum albumin modified with a redox-active compound in the form of neutral red, predominantly in an amount of 95.0-97.0 wt.%; inclusions of nanomaterial in the form of single-wall carbon nanotubes (2) placed in said polymer matrix in an amount of 1.0-2.0 wt.%.

[0037] - and immobilized in the said polymer matrix inclusions of a biorecognition component in the form of the lactate oxidase enzyme (3) in an amount of 2.0-3.0 wt.%.

[0038] To form the spatial structure of the polymer matrix (4) in the form of a hydrogel, bovine serum albumin is used in an amount of 3.7-5.0 wt.% (preferably 3.7 wt.%), a potassium-sodium phosphate buffer solution with a pH of 6-8, which also serves to regulate the pH of the mixture, in an amount of 30.0-50.0 wt.%, a mediator in the form of a saturated solution of neutral red in an amount of 5.0-7.0 wt.%, as well as a bifunctional crosslinking agent in the form of a solution of glutaraldehyde in an amount of 7.5-9.5 wt.%.

[0039] The claimed device includes a working electrode (1), the working surface of which is modified (coated) with the above-described composite material corresponding to the claimed invention. A printed graphite electrode or an inert metal electrode may be used as the working electrode (1).

[0040] Below are examples of obtaining the claimed group of inventions.

[0041] Example 1. In a test tube, 50 μl of potassium-sodium phosphate buffer solution with a pH of 6-8, 10 μl of a suspension of single-wall carbon nanotubes, 5 μl of a saturated solution of neutral red mediator, and the enzyme lactate oxidase (concentration 20 U / mg, volume 20 μl) are added to 0.0035 g of bovine serum albumin. The resulting mixture is shaken for 2 minutes. Then, 7.5 μl of a 25 wt% glutaraldehyde solution is added to the resulting mixture and shaken for no more than 30 seconds before applying it to a graphite printed electrode. Next, 3 μl of the resulting mixture is applied to the working surface of the graphite printed electrode and left until completely dry. As a result, a composite material is obtained with the following ratio of components: polymer matrix (4) - 97.0 wt.%, inclusions of single-wall carbon nanotubes (2) - 1.0 wt.% in terms of dry matter, inclusions of the lactate oxidase enzyme (3) - 2.0 wt.% in terms of dry matter.

[0042] Example 2. In a test tube, 30 μl of potassium-sodium phosphate buffer solution with a pH of 6-8, 20 μl of a suspension of single-wall carbon nanotubes, 7 μl of a saturated solution of neutral red mediator, and the enzyme lactate oxidase (concentration 20 U / mg, volume 30 μl) are added to 0.005 g of bovine serum albumin. The resulting mixture is shaken for 2 minutes. Then, 9 μl of a 25 wt% glutaraldehyde solution are added to the resulting mixture and shaken for no more than 30 seconds before applying it to a graphite printed electrode. Next, 3 μl of the resulting mixture is applied to the working surface of the graphite printed electrode and left until completely dry. As a result, a composite material is obtained with the following ratio of components: polymer matrix (4) - 95.0 wt.%, inclusions of single-wall carbon nanotubes (2) - 2.0 wt.%, inclusions of the lactate oxidase enzyme (3) - 3.0 wt.%.

[0043] Example 3. In a test tube, 40 μl of potassium-sodium phosphate buffer solution with a pH of 6-8, 15 μl of a suspension of single-wall carbon nanotubes, 6 μl of a saturated solution of neutral red mediator, and the enzyme lactate oxidase (concentration 20 U / mg, volume 25 μl) are added to 0.004 g of bovine serum albumin. The resulting mixture is shaken for 2 minutes. Then, 8 μl of a 25 wt% glutaraldehyde solution are added to the resulting mixture and shaken for no more than 30 seconds before applying it to a graphite printed electrode. Next, 3 μl of the resulting mixture is applied to the working surface of the graphite printed electrode and left until completely dry. As a result, a composite material is obtained with the following ratio of components: polymer matrix (4) - 96.0 wt.%, inclusions of single-wall carbon nanotubes (2) - 1.5 wt.%, inclusions of the lactate oxidase enzyme (3) - 2.5 wt.%.

[0044] The operating principle of the claimed group of inventions is as follows.

[0045] A potentiostat, a measuring cuvette (e.g., 5 ml), a magnetic stirrer, and a laptop are used for measurements. The device is placed in the measuring cuvette, a sodium-potassium phosphate buffer solution with a pH of 6-8 is added, the magnetic stirrer is turned on, and the background current in the measuring cuvette is recorded. A CS150 potentiostat (Contest, China), connected to a working electrode and a personal computer, can be used to record measurement results. The physiological fluid sample to be analyzed, which can be human blood or sweat, is then introduced. Measurements are performed at room temperature and an operating potential of -550 mV, due to the oxidation-reduction properties of the redox-active compound used in the composition—neutral red. After each measurement, the measuring cuvette is rinsed with a phosphate buffer solution with a pH of 6.8 in a volume equal to the volume of the measuring cuvette.

[0046] In Examples 1-3, a graphite printed electrode was used as the working electrode of the claimed device. The signal of the graphite printed electrode (1) is the dependence of the current strength (nA) on time (s) (see Fig. 3). Next, the amplitude of the change in current strength after introducing the sample into the measuring cuvette is calculated (biosensor response, AI, nA). The lactate content in the sample is determined using a pre-built calibration curve shown in Fig. 4. The calculation is carried out as follows: the lactate content in the sample is determined using the equation describing the linear portion of the calibration curve shown in Fig. 5 (y = 8.02 + 0.36, where x is the lactate concentration, mmol / dm 3 , y is the biosensor response, nA). Examples of the results of calculating the lactate concentration 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 lactate concentration in physiological fluids using the claimed group of inventions

[0048] The main characteristics of the claimed device for the quantitative assessment of lactate content in physiological fluids and the device according to the closest analogue are shown in Table 2.

[0049] Table 2. Comparative characteristics of devices for quantitative assessment of lactate content in physiological fluids

[0050] Thus, the claimed composite material and device for quantitative assessment of lactate content in physiological fluids make it possible to determine the lactate content of elevated concentrations (up to 57 mM) with a single measurement duration of no more than 2 minutes, which ensures a non-invasive express analysis of the quantitative assessment of lactate content in physiological fluids without dilution.

Claims

CLAUSES OF THE INVENTION 1. A composite material for a device for quantitatively assessing the lactate content in physiological fluids, comprising a polymer matrix (4), inclusions of a nanomaterial and inclusions of the lactate oxidase enzyme (3) immobilized in the polymer matrix (4), wherein the polymer matrix (4) contains a polymer modified with a redox-active compound, characterized in that bovine serum albumin is used as the polymer, neutral red is used as the redox-active compound, covalently bound to said polymer, and the nanomaterial is single-walled carbon nanotubes (2), wherein the composite material contains components in the following ratio, by weight. %: inclusions of single-walled carbon nanotubes - 1.0-2.0; inclusions of the lactate oxidase enzyme - 2.0-3.0; polymer matrix - the rest.

2. A composite material for a device for quantitatively assessing the lactate content in physiological fluids according to claim 1, characterized in that the composite material is obtained by mixing bovine serum albumin, potassium-sodium phosphate buffer solution, a suspension of single-wall carbon nanotubes, a saturated solution of neutral red, the enzyme lactate oxidase, and a solution of glutaraldehyde.

3. A device for quantitatively assessing the lactate content in physiological fluids, comprising a working electrode (1), characterized in that the surface of the working electrode (1) is coated with a composite material according to any of paragraphs 1-2.

4. A device for quantitatively assessing the lactate content in physiological fluids according to paragraph 3, characterized in that a graphite printed electrode or an inert metal electrode is used as the working electrode (1).

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