RU2800949C1 - METHOD FOR GENERATING HYDROGEN PEROXIDE BASED ON ZnO2 NANO- AND/OR MICROPARTICLES FOR APPLICATION IN SPECTROPHOTOMETRIC AND LUMINESCENT ANALYSIS WITH PARTICIPATION OF PEROXIDASE - Google Patents

Abstract

FIELD: analytical chemistry.

SUBSTANCE: present invention relates to compositions and kits for use in spectrophotometric (including colorimetric) and luminescent (fluorescent, chemiluminescent, bioluminescent) analysis, including bioanalytical, biosensor and medical technologies. It disclosed the use of nano- and/or microparticles of ZnO₂ to generate hydrogen peroxide for use in systems with peroxidase for spectrophotometric and luminescent detection and/or determination of both the peroxidase itself and its substrates. The invention also describes a method for generating hydrogen peroxide based on nano- and/or microparticles of ZnO₂ for use in spectrophotometric and luminescent analysis involving peroxidase. The reproducibility of results is at least 99% in the case of the spectrophotometric method of analysis and at least 98% in the case of the luminescent one, and the time of a single analysis is less than 7 minutes, which in the case of a 96-well plate is equivalent to the analysis time of 1 sample of less than 5 s. The rate of the reaction of enzymatic oxidation of indicator substrates is 30% higher when using ZnO₂ nanoparticles than when hydrogen peroxide is introduced from outside.

EFFECT: possibility of rapid determination of a wide range of biologically active substances, as well as peroxidase by enzyme activity, which is controlled by the accumulation of coloured or luminescent oxidation products of indicator substances (TMB, ABTS, FDA).

16 cl, 7 dwg, 2 tbl, 4 ex

Description

The field of technology to which the invention belongs

The present invention relates to analytical chemistry, in particular, to compositions and kits for use in spectrophotometric (including colorimetric) and luminescent (fluorescent, chemiluminescent, bioluminescent) analysis, including bioanalytical, biosensor and medical technologies. The scope of this development is the creation of bioanalytical and biosensor systems, analytical studies, medical diagnostics, enzyme immunoassay and DNA analysis (including through labeling with labels based on peroxidases), determination of enzyme-dependent biologically active substances. The invention can also be used to create biosensors based on oxidase enzymes that require hydrogen peroxide as a substrate.

State of the art

The generation of hydrogen peroxide inside the reaction medium is an important task for the development of new and improvement of existing methods of qualitative and quantitative analysis based on the use of enzymes, including as a label, in biology, medicine, and clinical diagnostics. The use of hydrogen peroxide makes it possible to effectively combine biochemical and physicochemical reactions when creating biosensors based on oxidase enzymes, which are widely used in enzyme biosensors, including as a label, and reactions catalyzed by them to determine biologically active substances due to their binding to specific ligands. .

Such techniques are based on the ability of biological molecules (receptors, antibodies, nucleic acids, enzymes) to bind with a high degree of specificity to the corresponding counterpart of the partner ligand. They are widely used in the determination of biologically active substances, and have become one of the main tools in biological research and medical diagnostics.

Sensor systems based on enzymes both as a recognizing ligand and as a label are the most widely used in modern biochemical analysis.

One of the classes of enzymes that have received wide commercial distribution, in particular, in enzyme immunoassay (ELISA or ELISA, Enzyme-Linked ImmunoSorbent Assay), determination of oligonucleotides, nucleic acid hybridization methods, is peroxidase, which has specificity for a wide group of compounds with different properties and structures. Peroxidase is a two-substrate enzyme and catalyzes 2 types of related reactions: (a) reduction of hydrogen peroxide to water and (b) oxidation of the second substrate, which include aromatic amines, phenolic compounds, and other substances.

The content of the above compounds is estimated by the activity of the peroxidase enzyme, which, in turn, is determined by the accumulation of absorbing or luminescent oxidation products of indicator substances - peroxidase substrates, for example, such as 3,3',5,5'-tetramethylbenzidine (TMB), 2 ,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and o-phenylenediamine (PDA), but the range of substrates is not limited to the examples listed. The choice of substrates depends on the detection method (spectrophotometric or luminescent), and also on the sensitivity requirements of the assay.

Despite numerous advantages and widespread use of peroxidase in biosensor technologies and bioanalytical applications, systems based on it have a key drawback. They require the addition of the main peroxidase substrate, hydrogen peroxide or H ₂ O ₂ , from outside, since it is extremely difficult to include this reagent in the composition of the sensor device at the stage of its manufacture. In addition, hydrogen peroxide is unstable during storage, which requires the use of various methods for its stabilization: using urea peroxide [Jones RM Stable peroxide substrate formulation // European Patent Office. Vol. 0 279 614], tin halides, using a stabilizer system consisting of a phosphonic acid chelating agent, a tin stabilizer, and an aromatic chelating compound [Wang X., Genco KR Stabilization of alkaline hydrogen peroxde // United States Patent. 2007 Vol. 2, no. 12]. At the same time, the described stabilization methods do not provide the stability of sensors necessary for routine analysis, and also adversely affect the sensitivity and reproducibility of the analytical signal [Kornienko VL et al. The Prospects of the in situ and ex situ Use of Aqueous Solutions of Hydrogen Peroxide Electrogenerated from Oxygen // Russ. J. Electrochem. © Pleiades Publishing, 2020. Vol. 56, no. 5. P. 427-441]. An alternative way to solve this problem can be the development of a method for generating hydrogen peroxide directly in the reaction mixture, as part of a bioanalytical system or a sensor device.

The prior art method of internal generation of hydrogen peroxide from oxygen on gas diffusion electrodes (GDE) from carbon black [VL Kornienko, GA Kolyagin, GV Kornienko and TA Kenova “ The prospects of the in-situ and ex-situ use of aqueous solutions of hydrogen peroxide electrogenerated from oxygen ”, Russian Journal of Electrochemistry, 2020, 56, 427-441]. The use of GDE based on technological carbon A-437E (acetylene black) and mesostructured carbon TsMK-3 makes it possible to obtain an H ₂ O ₂ solution with a concentration above 3 M, which can be used with high efficiency both in the indirect electrosynthesis of important organic and inorganic target products, and in the destruction of organic and inorganic pollutants present in wastewater of various origins.

However, the known solution involves the use of a carbon black electrode, which, due to the color of the tool, will complicate detection. In this case, during electrolysis, secondary oxidation and destruction of molecules of chromogenic substrates is possible, which interferes with the controlled oxidation process by the generated hydrogen peroxide. Finally, the generation of hydrogen peroxide with an efficiency of 96% by this method takes 30 min, which worsens the accuracy of the analysis, its reproducibility, and rapidity. The latter limits the use of such a system in the determination of analytes unstable in time.

The prior art method of photocatalytic internal generation of hydrogen peroxide from peroxo compounds [Lanchuk Y. v. et al. “ Towards sustainable diagnostics: replacing unstable H2O2 _by photoactive TiO2 _in testing systems for visible and tangible diagnostics for use by blind people ”, RSC _Advances , Royal Society of Chemistry, 2018. Vol. 8, No. 66. P. 37735-37739]. The authors of the work propose to replace unstable H ₂ O ₂ with stable TiO ₂ as an alternative source of acrylamide radical polymerization initiators in order to create a visual and tactile portable sensor for detecting biologically important molecules (DNA, RNA, ATP). Hydrogen peroxide is generated directly in the reaction medium by the reduction of air oxygen dissolved in an aqueous solution by photogenerated electrons formed during UV irradiation of an aqueous solution of TiO ₂ nanoparticles. TiO ₂ destroys the structure of water under the influence of irradiation, which leads to the formation of a high concentration of reactive oxygen species, including hydrogen peroxide, as well as free radicals.

However, this method is non-specific and non-selective: the UV irradiation process will oxidize most of the components of the system, not just the desired analytes. In this case, UV radiation can also lead to the destruction of some of the organic molecules involved in the process. In addition, the amount of hydrogen peroxide obtained by this method (at the level of units of micromolar concentrations) is not enough to carry out the reaction of peroxidase oxidation of indicator substances - the second substrates of the enzyme.

The prior art method of electrolysis of water to produce hydrogen peroxide as needed: hydrogen and oxygen are mixed in the electrolyzer, and then their mixture in water is fed into the reactor to obtain the target reagent [Patent KR20090014396A, 10.02.2009]. The solution is produced without the need to add additional reagents. The present invention provides for the production of an aqueous solution of hydrogen peroxide as a stream in series or in parallel with a standard plumbing. Gaseous hydrogen and oxygen obtained from tap water in the electrolytic cell are sent to a reactor in communication with the electrolyzer and located between the electrolyzer and the outlet, where they then react in the presence of a catalyst (based on platinum, palladium, ruthenium, rhodium, iridium, osmium and gold) to form hydrogen peroxide in the aqueous phase, which is then discharged through the outlet. The hydrogen peroxide detection sensors in the present invention include spectroscopic methods, such as ultraviolet or infrared spectroscopy methods, as well as potentiometric methods.

However, this method of generating hydrogen peroxide is not suitable for creating compact biosensor and bioanalytical systems. In addition, as shown above, electrochemical generation requires a certain amount of time to accumulate the required amount of hydrogen peroxide and may be accompanied by the destruction of organic molecules and biological objects.

Closest to the claimed is a method for generating hydrogen peroxide using calcium and magnesium peroxide [Waite, AJ, Bonner, JS, Austenite, R. " Kinetics and stoichiometry of oxygen release from solid peroxides ". Environ. Eng. sci. 1999, 16, 187-199]. This method consists in introducing solid particles of calcium and magnesium peroxide into an aqueous solution of alkalis with a pH of 8 to 10, as a result of a surface reaction, hydrogen peroxide is released in an amount of up to 50% of that contained in the system.

These solid forms of peroxides are widely used for environmental purposes due to their stability in high pH aqueous solutions. The stability of calcium and magnesium peroxides has been reported, which can be compared to metal hydroxide, which creates a growing mass transfer barrier, which slows down the further release of hydrogen peroxide, and also helps to maintain a high pH in the layers near the peroxo compound.

However, the transport barrier formed during the precipitation of hydroxide in nanoparticles is too thin to noticeably slow down the release of hydrogen peroxide and allow the process to be carried out in a more controlled manner. Especially in the region close to neutral pH, as well as at acidic pH, rapid dissolution is preferable due to the reaction MO _{2 (solid)} + 2H ⁺ _(liquid) → M ²⁺ _(liquid) + H ₂ O _{2 (liquid)} .

In addition, this method is limited to use only in alkaline conditions, since calcium and magnesium peroxides release hydrogen peroxide only at pH = 9, which is incompatible with the region of maximum activity of horseradish peroxidase (pH ~ 6) and is not suitable for the purposes of analysis. where it applies.

Thus, there is a need to develop a method for generating hydrogen peroxide, which, firstly, is based on inexpensive materials that are stable during long-term storage (pure hydrogen peroxide is not stable), and secondly, proceeds at a pH close to the pH of the maximum activity of peroxidases (improvement of kinetic parameters of biochemical reactions), thirdly, it does not have significant absorption and/or fluorescence in the near ultraviolet and visible parts of the spectrum (requirements for spectrophotometric and fluorimetric detection).

The technical problem to be solved by the claimed invention is the development of a method for internal ( in-situ ) generation of H ₂ O ₂ on demand (on-demand) by adjusting the pH of the system with a buffer solution for direct use in sensor systems that require the presence of hydrogen peroxide for their work, in particular, in systems that contain peroxidase. To solve this problem, it is proposed to use ZnO ₂ nanoparticles that are stable both during storage and when preparing a solution with a pH above 7.

The claimed invention eliminates the above disadvantages of analogues.

Disclosure of invention

The technical result of the invention is the possibility of rapid determination of a wide range of biologically active substances, as well as peroxidase by enzyme activity, which is controlled by the accumulation of colored or luminescent oxidation products of indicator substances (TMB, ABTS, FDA). This achieves reproducibility of results of at least 99% in the case of the spectrophotometric method of analysis and at least 98% in the case of the luminescent one, and the time of a single analysis is less than 7 minutes, which in the case of a 96-well plate is equivalent to the analysis time of 1 sample of less than 5 s. The rate of the reaction of enzymatic oxidation of indicator substrates is 30% higher when using ZnO ₂ nanoparticles than when hydrogen peroxide is introduced from outside.

At the same time, nano- and microparticles of ZnO ₂ are stable for a long time (more than a year) when stored in air without access to CO _2. In its pure form, hydrogen peroxide is not stable during long-term storage, therefore, special substances are used to stabilize it, which, including , can influence the course of peroxidase oxidation processes. Thus, the use of long-term storage-stable ZnO ₂ particles makes it possible to improve the reproducibility of the results obtained and to increase the rate of the peroxidase oxidation reaction.

The technical result is achieved by using nano- and/or microparticles of ZnO ₂ to generate hydrogen peroxide for use in systems with peroxidase for spectrophotometric and luminescent detection and/or determination of both the peroxidase itself and its substrates. Nano- and/or microparticles of ZnO ₂ are used in the form of a suspension in both aqueous and non-aqueous solvents, on the surface or in the volume of polymer films.

Also, the technical result is achieved by a method for generating hydrogen peroxide based on nano- and / or microparticles of ZnO ₂ for use in spectrophotometric and luminescent analysis with the participation of peroxidase, including the addition of an enzyme solution in a buffer solution with pH < 6 in an amount that ensures the peroxidation reaction, the introduction a sample containing an indicator substrate, followed by adding a pre-prepared suspension of zinc peroxide nanoparticles to the resulting mixture, after which the detection and determination of peroxidase activity and/or the content of oxidized oxidation products of indicator substrates is carried out by the spectrophotometric and/or fluorimetric method in a cuvette and/or microplate. In this case, the qualitative and quantitative determination of peroxidase is carried out by a spectrophotometric and/or fluorimetric method. As an indicator substrate, substances are used - dyes or biologically active substances that are substrates of peroxidase and change their color during the oxidation reaction, for their spectrophotometric and / or fluorometric determination, and substances selected from the group including TMB, FDA are used as a dye. , ABTS. As biologically active substances, ascorbic acid, flavonoids (such as but not limited to epicatechin and quercetin), vitamin B1 (thiamine), hydroperoxides of various structures, phenothiazines, catecholamines and their metabolites are used. As a peroxidase, peroxidase from horseradish roots, soybeans, peanuts, tobacco leaves, sweet potato tubers and oil palm leaves is used, while this does not limit the use of peroxidases of fungi and animal origin, including humans. Peroxidase is contained in an amount of 0.05 to 1 nM. As a buffer solution, a buffer with a pH in the range of 5-6 units is used, including a phosphate buffer, a phthalate buffer solution, an acetate buffer solution. To determine peroxidase, signals of optical density and intensity of luminescence, excitation of luminescence in the sample are recorded from 5 to 7 minutes, and when a maximum is detected in the characteristic range for the analyzed analyte, a conclusion is made about its presence in the sample. The conclusion about the presence and quantity of peroxidase in the sample is made when a maximum is detected in the absorption spectra of its oxidized substrate 3, 3',5, 5'-tetramethylbenzidine (TMB) in the range of 370-380 nm and 650-660 nm in the case of the formation of an intermediate product of TMB oxidation (meriquinoid complex); 450-460 nm in the case of complete oxidation of TMB to the final product (quinonediimine, CDI). The conclusion about the presence and quantity of peroxidase in the sample, including as a label, is made when a maximum is detected in the absorption spectra of its oxidized substrate o-phenylenediamine (PDA) in the range and 420-450 nm in the case of peroxidation of PDA to the final product (2 ,3-diaminophenazine, DAP). The conclusion about the presence and quantity of peroxidase in the sample, including as a label, is made by detecting a maximum in the absorption spectra of its oxidized substrate 2,2'-azino-bis-(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS) in the range 410-420 nm in the case of ABTS peroxidation with the formation of a radical cation. The conclusion about the presence and quantity of peroxidase in the sample, including as a label, is made by detecting the maximum luminescence intensity in the emission spectra of its oxidized substrate o-phenylenediamine (PDA) in the range from 560 to 580 nm. To obtain a luminescence signal (2,3-diaminophenazine, DAP), the sample is irradiated with monochromatic radiation at a wavelength of 430 nm.

Brief description of the drawings

The invention is illustrated by drawings, where:

In FIG. 1 is a schematic representation of the external H ₂ O ₂ addition process (upper panel) and in-system generation (lower panel) in the sample volume.

In FIG. Figure 2 shows the scheme of the peroxidation reaction of the considered classical peroxidase substrates: TMB, FDA, and ABTS.

In FIG. Figure 3 shows the pH dependences of the optical density of the products of peroxidation of these chromogenic substrates, based on spectrophotometric data in the UV and visible ranges. A comparison was made of the efficiency of substrate oxidation with H ₂ O ₂ obtained by internal generation and with the addition of H ₂ O ₂ from outside. The positions in the figures indicate: 1 - the dependence of the optical density on the pH value in the case of the formation of CDI with an absorption maximum at λ = 450 nm and a meriquinoid complex with an absorption maximum at λ = 375/655 nm) at pH = 5.5, 8 and 10; 2 - dependence of the optical density on the pH value in the case of the formation of DAP with an absorption maximum at λ = 430 nm at pH = 5.5, 8, 10.

In FIG. 4 shows data comparing the kinetics of FDA oxidation catalyzed by peroxidase), using H ₂ O ₂ added from the outside, and H ₂ O ₂ obtained by the inventive method of internal generation by adjusting the pH of ZnO ₂ nanoparticles. The kinetic curves are plotted on the basis of the data of the luminescence spectra during PDA oxidation.

In FIG. Figure 5 presents data comparing the kinetics of the oxidation of ABTS catalyzed by peroxidase, using H ₂ O ₂ added from the outside, and H ₂ O ₂ obtained by the inventive method of internal generation by adjusting the pH of ZnO ₂ nanoparticles. The kinetic curves are plotted based on the absorption spectra of the ABTS oxidation products.

In FIG. Figure 6 shows the time dependences of the optical density of the products of peroxidation of substrates on the concentration of peroxidase under the conditions of internal generation of hydrogen peroxide, based on spectrophotometric data in the UV and visible ranges. The positions in the figures indicate: 1 - the dependence of the optical density on the concentration of peroxidase in the case of the formation of CDI with an absorption maximum at λ = 450 nm; 2 - dependence of the optical density on the concentration of peroxidase in the case of the formation of DAP with an absorption maximum at λ = 430 nm; 3 - dependence of the optical density on the concentration of peroxidase in the case of the formation of ABTS ^•+ with an absorption maximum at λ = 415 nm in the time period of 0-10 min.

In FIG. Figure 7 shows the time dependence of the luminescence intensity of the PDA peroxidation product under the conditions of internal generation of hydrogen peroxide on the peroxidase concentration in the time period of 0–10 min.

Implementation of the invention

Replacement of externally added ( ex-situ ) H ₂ O ₂ with ZnO ₂ nanoparticles generating hydrogen peroxide directly in the test system under the action of a buffer solution with pH < 6 improves the reproducibility of results in the determination of enzyme-dependent biologically active substances and increases the reaction rate peroxidase oxidation.

A method for internal generation of hydrogen peroxide using ZnO ₂ nanoparticles for determining biologically active substances by peroxidase activity includes mixing a sample containing an indicator substrate with a peroxidase solution and a colloidal solution of ZnO ₂ nanoparticles, followed by adding a buffer solution with pH < 6 in an amount that ensures the reaction peroxidation to form colored and/or luminescent products.

It is preferable to use peroxidase from horseradish roots, soybeans, peanuts, tobacco leaves, sweet potato tubers and oil palm leaves as peroxidase, while this does not limit the use of peroxidases of fungi and animal origin, including humans.

Among the stable metal peroxides, zinc peroxide ZnO ₂ is promising as a source of hydrogen peroxide because it is cheap to produce, has a long shelf life, and quantitatively releases H ₂ O ₂ at a pH below 6, which corresponds to the pH range of maximum peroxidase activity, according to the reaction:

Thus, ZnO ₂ nanoparticles are proposed to be used as a material for the intrasystem generation of H ₂ O ₂ as an alternative to adding H ₂ O ₂ from outside.

Nanoparticles were synthesized in accordance with the methodology presented in the publication [Wolanov Y. et al. Zinc Dioxide Nanoparticulates: A Hydrogen Peroxide Source at Moderate pH // Environ. sci. Technol. 2013. Vol. 47. P. 8769-8774]: 1.0 g (4.6 mmol) of zinc acetate dihydrate was dissolved in 12 ml of 1.1 wt. % hydrogen peroxide solution. 25 ml of 0.4M (10 mmol) was added slowly until a pH of 8-9 was reached. As a result, spherical aggregates of zinc peroxide with an average diameter of 30-40 nm were obtained according to the results of scanning electron microscopy.

In the case of spectrophotometric detection, the presence of a peroxidase substrate is required, which gives a colored compound during the reaction. To do this, the sample solution in a cuvette or a transparent microplate is placed in a spectrophotometer and optical density values are recorded in the range from 200 to 900 nm. The conclusion about the presence and quantity of peroxidase in the sample is made by detecting the maximum absorption of its oxidized substrate in the range: 370-380 nm and 650-660 nm in the case of the formation of an intermediate product of TMB oxidation (meriquinoid complex); 450-460 nm in the case of complete oxidation of TMB to the final product (quinonediimine, CDI); 410-420 nm in the case of formation of the radical cation ABTS ^•+ , as well as 420-450 nm in the case of FDA peroxidation to the final product (2,3-diaminophenazine, DAF).

In the case of luminescent detection (using FDA as an example), the sample is irradiated with monochromatic radiation with a wavelength in the range of 420–450 nm (in our case, 430 nm). The conclusion about the presence and amount of peroxidase in the sample is made by detecting the maximum intensity of the luminescence of its oxidized substrate 2,3-diaminophenazine in the range from 560 to 580 nm (to be more precise, 570 nm). Kinetic data were obtained by measuring fluorescence 1, 2, 3, 5, and 10 min after the start of the reaction.

To determine the quantitative content of analytes in the test samples, the intensity of the received signal is measured (spectrophotometric and luminescent determination), the conclusion about the quantitative content is made according to pre-calculated calibration dependencies.

The effectiveness of this approach has been demonstrated on classical peroxidase substrates: 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2'-azino-bis-(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS) (spectrophotometric) , as well as o-phenylenediamine (PDA) (spectrophotometric and luminescent determination). The system using this method of hydrogen peroxide generation is characterized by a low detection limit, high selectivity, a wide linear range of determined contents (concentrations) of peroxidase substrates, as well as high reproducibility and rapidity.

Obtaining absorbing and luminescent products of peroxidation of peroxidase substrates as an indicator of the efficiency of the internal generation of hydrogen peroxide (Fig. 1, 6, 7), includes the oxidation of the starting compounds in the presence of a biocatalyst - peroxidase of plant origin, in particular peroxidase from horseradish roots. For typical chromo and fluorogenic peroxidase substrates (TMB, ABTS, or PDA), schemes of such oxidation reactions are shown in Figs. 2.

The oxidation of the starting compounds is carried out due to the internal generation of hydrogen peroxide in the biosensor and/or bioanalytical systems instead of introducing a hydrogen peroxide solution from the outside, while the rate of the peroxidation reaction increases. This scheme of the invention greatly simplifies the mass express analysis procedure - 96 samples in 7 minutes, i.e. about 5 s per sample.

This method of generating hydrogen peroxide can be used both for detection in a cuvette (plastic, glass or quartz), glass plate, optical fiber, and with any solid or prefabricated polymer (for example, polystyrene, polypropylene, etc.) plates with any number of wells, of which the most applicable are 6, 12, 24, 48, 72, 96-well (but not limited to), suitable for spectrophotometric and luminescent analysis. At the same time, their chemical and thermal stability does not matter, since the analysis is carried out in an aqueous medium at room temperature.

The method for determining the products of peroxidation of peroxidase substrates described in the claimed invention includes the sequential introduction of reaction components: phosphate buffer solution (PBS), peroxidase solution, substrate solution (TMB, ABTS or FDA), solution of ZnO ₂ nanoparticles.

Preferably, the reaction components are introduced one by one in the following order:

• ZnO ₂ nanoparticles (100 - 1000 µM, dissolved in buffer solution)

• PBS (phosphate buffer solution; 0.1 - 1 M, pH 4.0 - 5.9)

• Peroxidase (0.5 - 1 nM dissolved in buffer solution)

After adding all the listed components, the reaction mixture is thoroughly mixed and the optical density or luminescence intensity is measured (in the case of the formation of a luminescent product) after 7 minutes, since this corresponds to the kinetic curve of the oxidation process reaching a plateau (Fig. 4, 5).

Figure 3 presents a comparison of the pH dependence of the signal (spectrophotometric and luminescent) when adding hydrogen peroxide from the outside and its internal generation using ZnO ₂ nanoparticles.

The initial recorded luminescence spectra were the dependence of the emission intensity (I) on the wavelength (λ). An additional mathematical processing of the intensity of the luminescent signal was used to construct the FDA oxidation kinetic curve: an integration algorithm with an exponential model was used to describe the kinetic curves of the enzymatically catalyzed peroxidation process.

The invention also makes it possible to reduce the time of a single analysis by 30%, which can be used in analytical studies, mass analyzes of biological fluids for medical diagnostic purposes, enzyme immunoassay and DNA analysis, biochemical studies to determine a wide range of analytes.

Qualitative and quantitative determination and conclusion about the presence of one or another peroxidase substrate is done by the presence of maxima in the absorption and emission spectra and their intensity, as described above.

In the present invention, the following designations and terms are adopted:

R - correlation coefficient,

λ _em - emission (emission) wavelength,

λ _ex - luminescence excitation wavelength,

PCH - peroxidase from horseradish roots,

FBI - phosphate buffer solution,

TMB - 3,3',5,5'-tetramethylbenzidine,

FDA - o-phenylenediamine,

HDI - quinonediimine,

DAF -2,3-diaminophenazine,

ABTS - 2,2'-azino-bis-(3-ethylbenzthiozolin-6-sulfonic acid),

ABTS ^•+ - radical cation ABTS.

Chemical symbols/abbreviations have their usual meanings: °C (degree(s) Celsius), nm (nanometer(s)), mm(millimeter(s)), min(minute(s)), s(second(s)) , µl (microliter (microliters)), M (mol (moles) per liter), L (liter (liters)), µl (microliter (microliters)), mg (milligram (milligrams)), nmol (nanomoles (nanomoles)) , nM (nanomoles (nanomoles) per liter), µM (micromoles (micromoles) per liter), mM (millimoles (millimoles) per liter), V (volts (volts)).

Nanoparticles - particles of material ranging in size from 0.1 to 100 nm, microparticles - particles ranging in size from 100 nm to 100 micrometers.

Indicator substrate - a substance that is a substrate of peroxidase and is capable of changing its color during the oxidation reaction, which makes it possible to detect and/or determine the given substance by spectrophotometric and/or fluorometric detection. Examples of indicator substrates are TMB, FDA, ABTS, but are not limited to them.

The following examples of specific implementation of the invention are provided to provide specialists in the art with a complete description of the conduct and application of the assay according to the invention, but do not limit the scope of the invention intended by the inventors.

All of the following reagents, cuvettes and plates are commercially available. All procedures, unless otherwise noted, were carried out at room temperature or ambient temperature, that is, in the range from 18 to 25°C; in the course of all experiments for the preparation of aqueous solutions used deionized water of high purity, purified using the installation "Milli-Q", "Millipore"; measurement of the pH of the buffer solution was carried out on a pH - millivoltmeter "Hanna Instruments" (Germany); To fix the time of the indicator reactions, a stopwatch manufactured by Agat (Russia) was used with an error of ±0.2 s. Absorption spectra in the UV and visible regions were recorded using a SPECTROstar Nano spectrophotometer (BMG Labtech, Germany) with an accuracy of ±0.003 opt. units Data collection and spectra processing were performed using the SPECTROstar Nano MARS software (BMG Labtech, Germany); luminescence intensity and emission spectra were recorded on a Cary Eclipce spectrofluorimeter (Agilent Technologies, USA); for measurements in solution, a white 96-well polystyrene plate (Costar, USA) was used (cell volume 300 μl); ultrasonic treatment was carried out using an ultrasonic bath "Sapphire 680" (PKF Sapphire, Russia); preparations were weighed with an accuracy of ±0.02 mg on an analytical balance "Discovery", "OHAUS"; Precise volumes of liquids were collected using Eppendorf automatic pipettes with volume ranges of 2-20, 10-100, 20-200, 100-1000 and 500-5000 µl.

Example 1 Definition peroxidase from horseradish roots by the reaction of TMB peroxidation under conditions of internal generation of hydrogen peroxide using spectrophotometric detection.

The method for determining the concentration of peroxidase, at which the highest efficiency of TMB peroxidation is achieved, involves obtaining an optical signal during the formation of a colored product (CDI).

204 µl of 0.1 M PBS with pH = 5.5, 36 µl of a 50 µM alcoholic solution of TMB, and 30 µl of a 100 µM solution of zinc peroxide nanoparticles in PBS are sequentially injected into a 96-well transparent polystyrene plate. Then, from 12 to 60 μl of a PQC solution in PBS was injected, which made it possible to vary the concentration in the range of 0.05 - 1 nM (Fig. 6). After that, the reaction mixture was thoroughly mixed and the optical density was measured regularly every minute up to 10 minutes. The control experiment was carried out similarly to the method described above, except that PBS was administered instead of PCC (Table 1).

Example 2 Definitionperoxidase from horseradish roots by the FDA peroxidation reaction under conditions of internal generation of hydrogen peroxide using spectrophotometric detection.

The analysis was carried out analogously to example 1 with the difference that 32 µl of a 50 µM aqueous solution of FDA was added to the system (Fig. 6, Table 1).

Example 3 Definition peroxidase from horseradish roots by the ABTS peroxidation reaction under conditions of internal generation of hydrogen peroxide using spectrophotometric detection.

The analysis was carried out analogously to example 1 with the difference that 48 µl of a 50 µM aqueous solution of FDA was added to the system (Fig. 6, Table 1).

Example 4 Definition peroxidase from horseradish roots by the FDA peroxidation reaction under conditions of internal generation of hydrogen peroxide using luminescent detection

The method for determining the peroxidase concentration at which the highest efficiency of FDA peroxidation is achieved involves obtaining a fluorescence signal and its further evaluation in the formation of a colored product (DAF).

Into a 96-well white polystyrene plate, 207 μl of 0.1 M PBS pH = 5.5, 32 μl of 50 μM PDA aqueous solution, and 30 μl of 100 μM zinc peroxide nanoparticles solution in PBS are sequentially injected. Then, from 12 to 60 μl of the PQC solution in PBS was injected, which made it possible to vary the concentration in the range of 0.05 - 1 nM (Fig. 7). After that, the reaction mixture was thoroughly mixed and the luminescence intensity was measured regularly for 10 min. The control experiment was carried out similarly to the method described above, except that PBS was administered instead of PCC (Table 2).

Table 1. Time dependence of optical density on peroxidase concentration in peroxidation reactions of the considered chromogenic substrates (TMB, FDA, ABTS) with the formation of colored products of quinonediimine (CDI), 2,3-diaminophenazine (DAF), and radical cation ABTS (ABTS ^•+ ) With _PKH , nM Optical density
(TMB, λ = 450 nm) Optical density
(FDA, λ = 430 nm) Optical density
(ABTS, λ = 415 nm) 0 min 3 min 5 minutes 7 min 10 min 0 min 3 min 5 minutes 7 min 10 min 0 min 3 min 5 minutes 7 min 10 min 1.00 0.77 1.81 2.13 2.35 2.51 0.19 0.31 0.37 0.41 0.43 0.13 0.22 0.28 0.33 0.34 0.50 0.36 0.74 0.86 0.94 0.99 0.16 0.22 0.27 0.30 0.32 0.10 0.14 0.17 0.20 0.21 0.10 0.08 0.10 0.09 0.08 0.09 0.07 0.07 0.08 0.08 0.08 0.07 0.07 0.08 0.08 0.08 0.05 0.07 0.08 0.08 0.08 0.08 0.07 0.07 0.08 0.07 0.07 0.07 0.07 0.07 0.07 0.07

Table 2. Temporary dependence of fluorescence intensity on peroxidase concentration in the reaction of FDA peroxidation with the formation of 2,3-diaminophenazine (DAF)

With _PKH , nM Fluorescence intensity, o. e.
(FDA λ _em = 570 nm: λ _ex = 430 nm) 0 min 3 min 5 minutes 7 min 10 min 1.00 0.19 0.31 0.37 0.41 0.43 0.50 0.16 0.22 0.27 0.30 0.32 0.10 0.07 0.07 0.08 0.08 0.08 0.05 0.07 0.07 0.08 0.07 0.07

Claims (16)

1. The use of nano- and/or microparticles of ZnO ₂ to generate hydrogen peroxide for use in systems with peroxidase for spectrophotometric and luminescent detection and/or determination of both the peroxidase itself and its substrates. 2. Application according to claim 1, characterized in that ZnO ₂ nano- and/or microparticles are used in the form of a suspension in both aqueous and non-aqueous solvents, on the surface or in the volume of polymer films. 3. A method for generating hydrogen peroxide based on nano- and/or microparticles of ZnO ₂ for use in spectrophotometric and luminescent analysis with the participation of peroxidase, including adding an enzyme solution in a buffer solution with pH < 6 in an amount that ensures the peroxidation reaction, introducing a sample, containing an indicator substrate, followed by adding a pre-prepared suspension of zinc peroxide nanoparticles to the resulting mixture, after which the detection and determination of peroxidase activity and/or the content of oxidized oxidation products of indicator substrates is carried out by the spectrophotometric and/or fluorimetric method in a cuvette and/or microplate. 4. The method according to claim 3, characterized in that the qualitative and quantitative determination of peroxidase is carried out by a spectrophotometric and/or fluorimetric method. 5. The method according to claim 3, characterized in that substances are used as an indicator substrate - dyes or biologically active substances that are substrates of peroxidase and change their color during the oxidation reaction, for their spectrophotometric and / or fluorometric determination. 6. The method according to claim 5, characterized in that substances selected from the group including TMB, FDA, ABTS are used as a dye. 7. The method according to claim 5, characterized in that ascorbic acid, flavonoids - such as epicatechin and quercetin, vitamin B1, hydroperoxides of various structures, phenothiazines, catecholamines and their metabolites are used as biologically active substances. 8. The method according to claim 3, characterized in that peroxidase from horseradish roots, soybeans, peanuts, tobacco leaves, sweet potato tubers and oil palm leaves is used as peroxidase, while this does not limit the use of peroxidases of fungi and animal origin, including the number of a person. 9. The method according to claim 3, characterized in that peroxidase is contained in an amount of from 0.05 to 1 nM. 10. The method according to claim 3, characterized in that a phosphate buffer, a phthalate buffer solution, an acetate buffer solution are used as a buffer solution. 11. The method according to claim 3, characterized in that for the determination of peroxidase from 5 to 7 minutes, signals of optical density and luminescence intensity, excitation of luminescence in the sample are recorded, and when a maximum is detected in the characteristic range for the analyte to be determined, a conclusion is made about its presence in the sample . 12. The method according to claim 6, characterized in that the conclusion about the presence and amount of peroxidase in the sample is made when a maximum is detected in the absorption spectra of its oxidized substrate 3, 3',5, 5'-tetramethylbenzidine (TMB) in the range of 370-380 nm and 650-660 nm in the case of the formation of an intermediate product of the oxidation of TMB (meriquinoid complex); 450-460 nm in the case of complete oxidation of TMB to the final product (quinonediimine, CDI). 13. The method according to claim 6, characterized in that the conclusion about the presence and amount of peroxidase in the sample, including as a label, is made when a maximum is detected in the absorption spectra of its oxidized substrate o-phenylenediamine (PDA) in the range and also 420- 450 nm in the case of FDA peroxidation to the final product (2,3-diaminophenazine, DAP). 14. The method according to claim 6, characterized in that the conclusion about the presence and amount of peroxidase in the sample, including as a label, is made when a maximum is detected in the absorption spectra of its oxidized substrate 2,2'-azino-bis-(3- ethylbenzthiozolin-6-sulfonic acid) (ABTS) in the range of 410-420 nm in the case of peroxidation of ABTS with the formation of a radical cation. 15. The method according to claim 6, characterized in that the conclusion about the presence and amount of peroxidase in the sample, including as a label, is made by detecting the maximum luminescence intensity in the emission spectra of its oxidized substrate o-phenylenediamine (PDA) in the range from 560 up to 580 nm. 16. The method according to claim 13, characterized in that to obtain a luminescence signal (2,3-diaminophenazine, DAP), the sample is irradiated with monochromatic radiation with a wavelength of 430 nm.