Method for producing rod-shaped magnetite particles for DNA extraction from whole blood

Abstract

FIELD: biotechnology.SUBSTANCE: used in diagnostic and research laboratories. A method for producing rod-shaped magnetite particles for isolating deoxyribonucleic acid (DNA) from whole blood is proposed, which includes producing Fe3O4 magnetite rods. At the first stage, particles of akagenite β-FeOOH are formed by hydrolysis of 0.57 M aqueous solution of FeCl3 at a temperature of 120–125 °C for 3 h. After completion of the reaction, the suspension is cooled to room temperature, the precipitate is separated by centrifugation at 10000 g for 10 min, washed with deionized water and ethanol and dried at 60 °C throughout the day. The resulting akagenite particles are dispersed in 20 ml of ethylene glycol using ultrasonic treatment at 100 W for 15 minutes. The resulting suspension is placed in an autoclave and subjected to hydrothermal treatment at 200 °C for 8 hours with the formation of the magnetite phase Fe3O4. Magnetite particles are separated with a magnet, washed successively with water and ethanol and dried at 60 °C within 24 hours. Then, a SiO2 shell is formed by dispersing a 50 mg sample of dried magnetite rods in 5 ml of deionized water. Dispersion is carried out in five cycles, each of which includes stirring at 300 rpm for 10 min and ultrasonic treatment at 45 W for 3 min. Isopropanol is added to the resulting suspension at a rate of 15 ml of isopropanol for every 5 ml of water, after which the mixture is stirred for 20 min at 300 rpm. Then, while stirring, 0.5 ml of a 25% NH4OH solution and 0.5 ml of tetraethoxysilane are added to the aqueous alcohol dispersion of particles for every 50 mg of rods. The formation of the SiO2 shell is carried out at room temperature for 20 hours with constant stirring. After incubation, the particles are purified by magnetic decantation with deionized water at least five times. The process of forming a silicon dioxide shell is repeated twice, and the purified rod-shaped particles of Fe3O4@SiO2 are dispersed in a 5% solution of polyethylene glycol with a molecular weight of 4000 Da. Fe3O4@SiO2-NH2 particles can also be obtained.EFFECT: increasinng the quantity and improving the purity of DNA isolated from whole blood by using magnetic particles with increased specific surface area and sorption capacity.2 cl, 12 dwg, 2 tbl, 3 ex

Description

[0001] The invention relates to the field of nanomaterials and biotechnology and is intended for isolating deoxyribonucleic acid (DNA) from blood. It can be used in diagnostic and research laboratories.

[0002] DNA isolation is an important step in the molecular diagnostics of microorganisms and infectious diseases, PCR diagnostics (polymerase chain reaction), sequencing, genomic studies, and forensic examination [1-4]. However, DNA is present in biological samples in relatively small quantities compared to other components such as proteins, polysaccharides, phospholipids, and metabolites, which can significantly interfere with its detection. Damage to the primary structure of DNA during purification often leads to decreased sensitivity, poor reproducibility, or complete loss of suitability of the material for subsequent procedures, such as PCR amplification or sequencing [5].

[0003] Considering the multicomponent nature of biological samples and the low DNA concentration, it is necessary to select the optimal purification method. Various approaches are used for DNA isolation: liquid-liquid extraction [6], liquid-liquid microextraction [7-8], solid-phase extraction [9], and magnetic solid-phase extraction (MSPE) [10-12].

[0004] Methods based on the use of magnetic nanomaterials have become the most widely used in practice because they require fewer organic solvents, are low-cost, easy to implement, and provide rapid results [13-14]. Magnetic nanoparticles (MNPs) used in MSPE can be rapidly collected under the influence of an external magnetic field, eliminating the centrifugation step, which causes shear forces and potential degradation of nucleic acids. Furthermore, the surface of MNPs is relatively easy to modify to improve sorption efficiency.

[0005] Various polymers are used to modify the surface of MNPs, such as polyacrylic acid

[15] , chitosan-polyaniline copolymer

[16] , polydopamine

[17] , polyethyleneimine

[18] , and others, as well as organosilicon compounds

[19] . Polymers and organosilicon compounds can additionally contain functional amino, hydroxyl, or carboxyl groups to increase selectivity [20-22]. Currently, a silicon dioxide-based shell is formed on the surface of MNPs for the isolation of nucleic acids, which increases their stability and sorption capacity.

[0006] In addition to the chemical composition of the coating, the size and morphology of the particles play an important role. Modern commercial magnetic particles (MP) for DNA extraction are typically spherical in shape and have the following structure: a magnetite (Fe3O4) core coated with silicon dioxide (SiO2), or porous SiO2 microspheres with incorporated magnetite nanoparticles [23-24]. Such particles typically have a size of 0.5-10 μm, a specific surface area of ​​10-70 m 2 / g and a loading capacity of 1-10 ng DNA per 1 mg of particles [25-28].

[0007] Spherical magnetic particles, although widely used for DNA extraction, have some drawbacks. Compared with particles of other shapes, they have a smaller specific surface area, which limits the number of available nucleic acid binding sites and reduces DNA yield [29–31]. Furthermore, weak magnetic anisotropy slows down the magnetic separation process at low field gradients, and the spherical shape impairs the distribution of magnetic forces in the suspension, which can lead to aggregation and uneven binding of biomolecules [32–34].

[0008] Rod-shaped magnetic particles are a promising alternative. Rod-shaped magnetic particles offer several advantages over spherical particles for DNA extraction from biological samples. Their elongated shape provides a larger specific surface area, providing a greater number of active sites for nucleic acid binding and increasing sorption efficiency. Their pronounced magnetic anisotropy facilitates faster and more complete particle collection under the influence of an external magnetic field, especially at low gradients. Furthermore, the rod-shaped particle shape facilitates particle orientation along the field lines, accelerating separation and washing processes. Taken together, these properties make rod-shaped magnetic particles promising for faster and more efficient DNA extraction [25-37].

[0009] During the analysis of the literature, no data were found on the production and use of magnetite rod particles coated with silicon dioxide (SiO2) and / or its amino derivatives (SiO2-NH2) for DNA extraction from biological samples.

[0010] A method for producing spherical or granular magnetic nanoparticles is known, in which magnetite (Fe3O4), maghemite (γ-Fe2O3), or manganese-zinc ferrite (MnZnFe2O4) are used as the magnetic core. The particles are coated with silicon dioxide (SiO2) or an oxide composite based on silicon and aluminum and are characterized by an average size of 0.1 to 0.5 μm (for SiO2) or 0.1 to 10 μm (for Si-Al). The resulting magnetic particles are intended for the extraction of DNA from Escherichia coli bacterial cells. The patent describes two methods for applying a silicon dioxide coating to the surface of magnetic particles. In the emulsion method (W / O), a sodium silicate solution (Na2SiO3) is added to a magnetite suspension, the mixture is emulsified in an organic solvent (n-hexane or xylene) using the non-ionic surfactant sorbitan monolaurate (Span 20), and then the emulsion is introduced into an ammonium salt solution.During neutralization, a layer of silicon dioxide (SiO2) is deposited on the surface of the particles. The resulting particles are washed, filtered, and dried at a temperature of 60-100°C for 20 hours. In the aqueous precipitation method, a suspension of magnetite (1-10% by weight) with the addition of sodium silicate (0.3-2% calculated as SiO2) is neutralized with hydrochloric acid (HCl) with constant stirring, resulting in the deposition of SiO2 directly on the surface of the particles. The resulting material is dried at 60-100°C and subjected to heat treatment at 200-800°C in an inert atmosphere (nitrogen or argon), which helps to improve crystallinity, increase magnetization, and increase the sorption capacity of the resulting particles

[38] .

[0011] The disadvantages of these methods include their multi-step nature, high energy consumption, and the use of toxic reagents. The emulsion method uses organic solvents (n-hexane, xylene), which are highly toxic and explosive. The aqueous precipitation method requires high-temperature heat treatment in an inert environment, significantly complicating the process. Furthermore, these methods require strict control of synthesis conditions and enhanced chemical safety measures, limiting their scalability and industrial application. The sorption capacity of the resulting particles is no more than 1-2 μg of DNA per 1 mg of magnetic material, which does not ensure high efficiency when working with biological samples.

[0012] A method is known for producing magnetic nanoparticles of magnetite of spherical shape, doped with copper, zinc and cobalt cations, with the general formula Co x Zn y Cu z Fe 3-х-y-zO4 (where x = 0.003-0.084; y = 0.003-0.084; z = 0.0465-0.093), coated with a silicon dioxide (SiO2) shell. The synthesis is carried out by coprecipitation of the corresponding Fe salts 2+ , Cu 2+ , Zn 2+ and Co 2+ at pH ≈ 9 using NaOH and triethanolamine. The resulting nanoparticles are dispersed in ethanol with the addition of tetramethylorthosiloxane (TMOS), polyvinyl alcohol and concentrated HCl used as a hydrolysis catalyst, resulting in the formation of an amorphous SiO2 shell. The resulting particles are dispersed in a solution containing a chaotropic agent (guanidine isocyanate, guanidine chloride, urea, thiourea), salts to increase ionic strength (NaCl, KCl, MgCl2) and nucleic acid co-precipitants (ethanol, isopropanol, polyethyleneglycol). The finished suspension of magnetic particles is characterized by a primary particle size of 20-40 nm, the possibility of controlled agglomeration (up to 1-1.4 μm) and a specific surface area of ​​215-310 m 2 / g. A feature of the sorbent is the controlled aggregation of particles, achieved by changing the ionic strength of the medium or the intensity of mechanical action (stirring, ultrasound)

[39] .

[0013] Despite the high specific surface area (up to 310 m 2 / g), the disadvantages of this solution include the complex chemical composition of the magnetic sorbent and the need for strict stoichiometry during synthesis, which complicates scaling and reduces the reproducibility of the process. Furthermore, the study lacks quantitative data on the sorption capacity of the particles, and DNA was isolated not from biological samples (blood, tissue cells, etc.), but from pre-prepared solutions of purified human genomic DNA and marker mixtures. This allowed us to evaluate only the selectivity of sorption by fragment length (more than 500 base pairs), but not the quantitative measurement of DNA extraction efficiency in complex biological systems.

[0014] The work chosen as a prototype presents spherical magnetic particles coated with SiO2 for DNA extraction. The method for coating the shell is similar to our proposed method for coating rod-shaped magnetic particles. Magnetic particles are introduced into a mixture of ethanol, deionized water, and ammonia, and ultrasonic dispersion is performed. Tetroethoxysilane (TEOS) is then added, and at room temperature and constant stirring, TEOS hydrolysis and SiO2 polycondensation on the surface of the magnetic cores occur in an alkaline ammonia medium

[40] .

[0015] The disadvantage of the particles is their lower sorption capacity of ≈ 2.18 μg DNA / mg particles when isolating DNA from an aqueous solution, which is more than 5-7 times less than in our proposed invention when isolating DNA from whole blood.

[0016] The aim of the present invention is to develop a technology for producing magnetite (Fe3O4) rod particles with a silicon dioxide-based shell, designed for the efficient extraction of DNA from whole blood.

[0017] The technical result of the invention is to increase the quantity and improve the purity of DNA isolated from whole blood by using rod-shaped magnetic particles coated with silicon dioxide (SiO2) and / or aminofunctionalized silicon dioxide (SiO2-NH2), which have an increased specific surface area (up to 54 m 2 / g) and sorption capacity (up to 16 μg DNA per mg of MP for Fe3O4).

[0018] The essence of the invention lies in the fact that a method for producing rod-shaped magnetite particles for isolating DNA from whole blood, consisting of obtaining Fe3O4 magnetite rods, forming at the first stage β-FeOOH akagenite particles by hydrolysis of 0.57 M aqueous solution of FeCl3 in an autoclave at a temperature of 120-125 ° C for 3 hours, after completion of the reaction, the suspension is cooled to room temperature, the precipitate is separated by centrifugation at 10,000 g for 10 minutes, washed with deionized water and ethanol and dried at 60 ° C for 24 hours, the resulting β-FeOOH akagenite particles are dispersed in 20 ml of ethylene glycol, using ultrasonic treatment with a power of 100 W for 15 minutes, the resulting suspension is placed in an autoclave and subjected to hydrothermal treatment at 200 ° C for 8 hours, after reduction, the color of the particles changes from orange to black, which indicates the formation of the magnetite phase Fe3O4, the finished particles of the rod form of magnetite are separated with a magnet,washed successively with water and ethanol and dried at 60°C for 24 hours, then a SiO2 shell is formed, a 50 mg sample of dried magnetite rods is dispersed in 5 ml of deionized water, dispersion is carried out for five cycles, each of which includes stirring at 300 rpm for 10 min and ultrasonic treatment with a power of 45 W for 3 min, 100% isopropanol is added to the resulting suspension at the rate of 15 ml of isopropanol for every 5 ml of water, after which the mixture is stirred for 20 min at 300 rpm, then, while stirring, 0.5 ml of a 25% NH4OH solution and 0.5 ml of tetraethoxysilane are introduced into the water-alcohol dispersion of particles for every 50 mg of rods, the reaction of the SiO2 shell formation is carried out at room temperature for 20 hours with constant stirring, at the end of the incubation of the particles cleaned by magnetic decantation with deionized water at least five times, the process of forming a silicon dioxide shell is repeated twice,The purified Fe3O4@SiO2 rod-shaped particles are dispersed in a 5% solution of polyethylene glycol with a molecular weight of 4000 Da for additional stabilization of the suspension. The Fe3O4@SiO2 rod-shaped particles with the formed first shell of silicon dioxide are dispersed in a water-alcohol solution, then 0.7 ml of acetic acid and 0.5 ml of N-[3-(trimethoxysilyl)propyl]ethylenediamine are added for every 50 mg of particles, incubation is carried out for 20 h at 300 rpm, then the Fe3O4@SiO2-NH2 particles are purified by magnetic decantation and dispersed in a 5% solution of polyethylene glycol with a molecular weight of 4000 Da.

[0019] Fig. (1-10) show the transmission electron microscopy (TEM) images and element distribution maps for the Fe3O4@SiO2, Fe3O4@SiO2-NH2, and Extra Gen Black (Raissol Bio) samples; Fig. 11 is a comparative study of the sorption capacity of Extra Gen black (Raissol), Fe3O4@SiO2, and Fe3O4@SiO2-NH2 magnetic particles in DNA extraction from human whole blood; Fig. 12 is an electrophoretic analysis of DNA extraction products in 0.8% agarose gel from blood using Extra gen black (Raissol), Fe3O4@SiO2, and Fe3O4@SiO2-NH2 magnetic particles; Table 1 presents the physicochemical characteristics of the particles; Table 2 shows the purity of the obtained DNA solution (A260 / A280, A260 / A230) and the sorption capacity of magnetic particles.

[0020] The advantage of the proposed invention is that the rod-shaped magnetic particles have a more developed porous surface compared to traditional spherical magnetic nanoparticles, resulting in an increased specific surface area. Furthermore, the presence of functional OH and / or NH2 groups on the surface enables interaction with phosphate ions within DNA molecules. These particle characteristics, in turn, ensure more efficient DNA sorption and recovery while maintaining its structural integrity and solution purity, making the process reproducible and suitable for routine analytical procedures.

[0021] Example 1. Description of the technology for producing rod-shaped magnetic particles (Fe3O4) surface-modified with silicon dioxide (SiO2) and / or amino-functionalized silicon dioxide (SiO2-NH2).

[0022] Method 1. In the first step, magnetite (Fe3O4) rods are obtained. First, akagenite (β-FeOOH) is synthesized by hydrolysis of a 0.57 M aqueous solution of FeCl3 in an autoclave at a temperature of 120-125°C for 3 h. After completion of the reaction, the suspension is cooled to room temperature, the precipitate is separated by centrifugation at 10,000 g (10 min), washed with deionized water and ethanol, and dried at 60°C for 24 hours. The resulting akagenite β-FeOOH particles are dispersed in 20 ml of ethylene glycol, using ultrasonic treatment at a power of 100 W for 15 min. The resulting suspension is placed in an autoclave and subjected to hydrothermal treatment at 200°C for 8 hours. After reduction, the particles' color changes from orange to black, indicating the formation of the magnetite phase Fe3O4. The resulting rod-shaped magnetite particles are separated with a magnet, washed successively with water and ethanol, and dried at 60°C for 24 hours.

[0023] In the second stage, the SiO2 shell was formed. A sample of dried magnetite rods (50 mg) was dispersed in 5 ml of deionized water. Dispersion was carried out for five cycles, each of which included stirring at 300 rpm for 10 min and ultrasonic treatment at 45 W for 3 min. 100% isopropanol was added to the resulting suspension at a rate of 15 ml of isopropanol for every 5 ml of water, after which the mixture was stirred for 20 min at 300 rpm. Then, with stirring, 0.5 ml of a 25% NH4OH solution and 0.5 ml of tetraethoxysilane (TEOS) for every 50 mg of rods were added to the aqueous alcohol dispersion of particles. The reaction of SiO2 shell formation was carried out at room temperature for 20 h with constant stirring. After incubation, the particles are purified by magnetic decantation with deionized water at least five times. The process of forming the silicon dioxide shell is repeated twice.Purified rod-shaped particles of Fe3O4@SiO2 are dispersed in a 5% solution of polyethylene glycol with a molecular weight (M. w ) 4 kDa for additional stabilization of the suspension.

[0024] Method 2 differs from Method 1 in that rod-shaped particles with a formed first shell of silicon dioxide Fe3O4@SiO2 are dispersed in a water-alcohol solution, then 0.7 ml of acetic acid and 0.5 ml of N-[3-(trimethoxysilyl)propyl]ethylenediamine (EDPS) are added for every 50 mg of particles. Incubation is carried out for 20 h at 300 rpm. Then the Fe3O4@SiO2-NH2 particles are purified by magnetic decantation and dispersed in a 5% solution of polyethylene glycol with a molecular weight (M w ) 4 kDa.

[0025] Thus, the proposed two-stage method ensures the production of monodisperse Fe3O4 rods modified with a shell of silicon dioxide (SiO2) or aminofunctionalized silicon dioxide (SiO2-NH2). The coating thickness and homogeneity can be controlled by the number of hydrolysis cycles of organosilicon compounds (TEOS, EDPS) and the ratio of reaction medium components.

[0026] Example 2. The following are the results of the analysis of the physicochemical characteristics of the obtained magnetic particles Fe3O4@SiO2, Fe3O4@SiO2-NH2, as well as commercial particles Extra Gen Black (Raissol Bio).

[0027] The particle size and morphology were studied using TEM. Micrographs of the Fe3O4@SiO2, Fe3O4@SiO2-NH2, and Extra Gen Black (Raissol Bio) samples are shown in Figs. (1-10), and a summary of the physicochemical characteristics is presented in Table 1.

[0028] Table 1. Physicochemical characteristics of particles

[0029] Fig. (1-10) Bright-field transmission electron microscopy images of Fig. (1, 4) - Fe3O4@SiO2; Fig. (2, 5) - Fe3O4@SiO2-NH2; Fig. (3, 6) - Extra Gen Black (Raissol Bio). Element distribution maps corresponding to the samples of Fig. 7 - Fe3O4@SiO2, Fig. (8, 10) - Fe3O4@SiO2-NH2 and Fig. 9 - Extra Gen Black (Raissol Bio), respectively.

[0030] The electrokinetic potential (ζ-potential) of the studied particles at pH = 7 was: -31 mV for Fe3O4@SiO2, +43 mV for Fe3O4@SiO2-NH2, and -34 mV for Extra Gen Black (Raissol Bio).

[0031] Morphological analysis showed that the developed Fe3O4@SiO2 and Fe3O4@SiO2-NH2 particles have a rod-shaped form with a length of 800±64 nm and a diameter of 40±9 nm, as well as a porous surface. Elemental analysis confirmed the uniform distribution of the silicon dioxide shell along the entire length of the rods (Fig. 7 and Fig. 8, respectively) and the presence of NH2 groups for Fe3O4@SiO2-NH2 (Fig. 10), indicating successful coating formation.

[0032] According to the TEM results, the commercial magnetic particles Extra Gen Black (Raissol Bio) are polydisperse, non-uniformly shaped silicon dioxide microparticles with an average size of 1831 ± 229 nm, containing spherical magnetic Fe3O4 nanoparticles with a diameter of 13 ± 4 nm inside. The presence of a silicon dioxide shell was also confirmed by the results of elemental analysis (Fig. 9).

[0033] Specific surface area (S, m 2 / g), as noted earlier, is one of the key parameters determining the efficiency of nucleic acid sorption on the surface of magnetic particles. To evaluate this indicator, the BET (Brunauer-Emmett-Teller) method was used, based on the analysis of nitrogen adsorption isotherms at 77 K, which allows for the quantitative determination of the specific surface area of ​​nano- and microparticles. The BET method is a generally accepted approach for characterizing porous and micro- and nanodispersed systems, including magnetic iron oxide nanoparticles and their SiO2 composites, used in biotechnology and sorption processes [41-45].

[0034] Calculations showed that the rod-shaped magnetic particles Fe3O4@SiO2 and Fe3O4@SiO2-NH2 have a specific surface area of ​​about 54 m 2 / g, while for commercial Extra Gen Black particles (Raissol Bio) this figure is about 2.5 m 2 / G.

[0035] Thus, the morphological features of the rod-shaped particles allow for an almost twentyfold increase in specific surface area compared to the spherical particles of Extra Gen Black (Raissol Bio). This difference is explained by their structural features: the rod-shaped magnetic particles have an elongated shape and a porous surface. Furthermore, the -OH and -NH2 functional groups on the particle surface ensure the binding and retention of DNA molecules, which is confirmed by subsequent experimental results on nucleic acid extraction.

[0036] Example 3. The following are the results of a study of the sorption capacity of the developed Fe3O4@SiO2, Fe3O4@SiO2-NH2 particles and commercial Extra Gen Black particles (Raissol Bio) when used to extract DNA from human whole blood samples.

[0037] To evaluate the efficiency and suitability of particles for DNA extraction, the sorption capacity (mass of nucleic acids per unit mass of magnetic particles) and the purity parameters of the DNA solution, expressed as the ratio of optical densities (for the corresponding wavelengths) A260 / A280 and A260 / A230, were determined. The DNA solution is considered pure and suitable for further analysis if the ratio A260 / A280 ≥ 1.7 and A260 / A230 is not less than 1.5

[46] .

[0038] Human blood stabilized with ethylenediaminetetraacetic acid (EDTA) was used as a biological sample. DNA extraction was performed according to the standard protocol included in the GM Blood M 2.0 reagent kit (Raissol Bio), followed by the use of the studied magnetic particles at the nucleic acid sorption stage. For each magnetic particle sample, five whole blood samples were prepared for DNA extraction (n=5). The results of determining the sorption capacity and purity of the isolated DNA solution using Fe3O4@SiO2, Fe3O4@SiO2-NH2, and Extra Gen Black particles (Raissol Bio) are presented in Table 2.

[0039] Table 2 - Purity of the obtained DNA solution (A260 / A280, A260 / A230) and the sorption capacity of magnetic particles.

[0040] Fig. 11 - Comparative study of the sorption capacity of magnetic particles Extra Gen black (Raissol), Fe3O4@SiO2, Fe3O4@SiO2-NH2 in the extraction of DNA from human whole blood.

[0041] The comparative analysis results showed that Extra Gen Black magnetic particles (Raissol Bio) demonstrated a sorption capacity of 6.43 ± 1.20 μg DNA / mg magnetic material (based on Fe3O4), respectively. While Fe3O4@SiO 2, Fe3O4@SiO2-NH2 were characterized by a higher sorption capacity - 11.20 ± 1.04 and 15.47 ± 0.62 μg DNA / mg, respectively, which is 74% and 141% higher compared to commercial Extra Gen Black particles (Raissol Bio).

[0042] The obtained values ​​of optical ratios A260 / A280 and A260 / A230 for all the studied samples were within the target ranges (≥ 1.7 and ≥ 1.5, respectively), which indicates sufficient purity of the isolated DNA solutions and the absence of protein and salt impurities that interfere with further analysis.

[0043] Thus, the study confirmed the effectiveness and practical suitability of the developed Fe3O4@SiO2 and Fe3O4@SiO2-NH2 magnetic rods for DNA extraction from human whole blood. The highest sorption capacity of Fe3O4@SiO2-NH2 particles is explained by the presence of amino groups capable of electrostatic interaction with DNA phosphate groups, ensuring stronger nucleic acid binding. This advantage, combined with the rods' morphological features, allows for the use of a smaller amount of starting biomaterial while maintaining a high DNA yield, improving the cost-effectiveness and reproducibility of the method.

[0044] Example 4. The following is an electrophoretic study of isolated DNA from human whole blood.

[0045] To determine the integrity of genomic DNA isolated from human blood, electrophoresis was performed in 0.8% agarose gel. The gel was prepared using 1× TBE buffer (TBE is a buffer solution containing a mixture of tris(hydroxymethyl)aminomethane, boric acid, and EDTA) with the addition of ethidium bromide intercalating dye (final concentration 0.5 μg / ml). DNA samples (120–150 ng) were mixed with 4× sample loading buffer (Gel Loading Dye, Evrogen, Russia) and applied to the wells of the gel. DNA length markers of 100+ bp and 1 kb (Eurogen, Russia) were used as molecular weight markers. Electrophoresis was carried out in the same buffer at 130 V for 40 min. Visualization of the results and acquisition of gel images were performed using the ChemiDoc Imaging System (Bio-Rad, USA).

[0046] Fig. 12 - Electrophoretic analysis of DNA extraction products in 0.8% agarose gel from blood using Extra gen black (Raissol), Fe3O4@SiO2 and Fe3O4@SiO2-NH2 magnetic particles. Lanes: 1 - DNA molecular weight marker (Marker 1); 2 - DNA isolated using commercial Extra Gen Black magnetic particles (Raissol Bio); 3 - DNA isolated using Fe3O4@SiO2 particles; 4 - DNA isolated using Fe3O4@SiO2-NH2 particles; 5 - DNA molecular weight marker (Marker 2).

[0047] The electrophoretic pattern revealed distinct diffuse bands in regions corresponding to high-molecular-weight DNA (lanes 2-4). Intense bands are observed in lanes 3 and 4, corresponding to Fe3O4@SiO2 and Fe3O4@SiO2-NH2 particles, indicating the presence of isolated DNA. Commercial Extra Gen Black particles (Raissol Bio) (lane 2) also exhibit a DNA band, but its intensity is somewhat lower. The absence of additional low-molecular-weight fragments or traces of degradation confirms that the magnetic particle-based extraction process does not have a destructive effect on the DNA structure.

[0048] Thus, a method for producing magnetic particles of rod-shaped Fe3O4 modified with a shell of silicon dioxide (SiO2) and / or aminofunctionalized silicon dioxide (SiO2-NH2) intended for DNA extraction from whole blood has been developed and experimentally validated. The proposed technology allows for the production of monodisperse structures with controlled morphology, a porous surface, and stable surface functionalization, as confirmed by the study results. The optimal combination of the rod shape and the chemical nature of the coating ensures an increase in the specific surface area (up to 54 m 2 / g) and sorption capacity (11.20 ± 1.04 and 15.47 ± 0.62 μg DNA / mg Fe3O4) for Fe3O4@SiO2 and Fe3O4@SiO2-NH2 particles compared to commercial particles (Extra Gen Black, Raissol Bio). Moreover, the purity of the isolated DNA (A260 / A280 ≈ 1.7; A260 / A230 ≥ 1.5) for the developed particles meets the standards of molecular biology research, and electrophoretic analysis confirmed a high degree of preservation of the native DNA structure. The proposed method can be scaled up and used for the production of domestic DNA extraction systems with high efficiency, reproducibility and compatibility with existing analytical protocols.

[0049] The main advantage of the invention is the combination of morphological and chemical characteristics of the particle coating, which ensures high-quality DNA extraction from whole blood. The developed magnetic rod-shaped particles Fe3O4@SiO2 and Fe3O4@SiO2-NH2 can be used as the basis for the creation of domestic DNA separation systems that are highly efficient, reproducible, and compatible with existing analytical protocols.

[0050] Compared to the known solution, the invention makes it possible to increase the quantity and improve the purity of DNA isolated from whole blood by using rod-shaped magnetic particles coated with silicon dioxide (SiO2) and / or aminofunctionalized silicon dioxide (SiO2-NH2), which have an increased specific surface area (up to 54 m 2 / g) and sorption capacity (up to 16 μg DNA per mg of MP for Fe3O4).

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[0098] Table 1

[0099] Fe3O4@SiO2 Fe3O4@SiO2-NH2 Extra gen black (Raissol Bio) Dimensions of rod-shaped particles (diameter / length), nm 40±9 / 800±64 40±9 / 800±64 - Size of spherical particles, nm - - 1831±229 ζ-potential, mV -31 +43 -34 Specific surface area, m2 / g 54 54 2,5

[0100] Table 2

[0101] A260 / A280 A260 / A230 Sorption capacity, μg (DNA) / mg (MP for Fe3O4) Fe3O4@SiO2 1.77 ± 0,09 1,51 ± 0,02 11,20 ± 1,04 Fe3O4@SiO2 -NH2 1,73 ± 0,05 1,57 ± 0,01 15.47 ± 0,62 Extra Gen Black (Raissol Bio) 1,81 ± 0,10 1,60 ± 0,05 6,43 ± 1,20

Claims

1. A method for producing rod-shaped magnetite particles for DNA extraction from whole blood, consisting of producing Fe3O4 magnetite rods, in the first stage β-FeOOH akagenite particles are formed by hydrolysis of 0.57 M aqueous FeCl3 solution in an autoclave at 120-125°C for 3 hours, after completion of the reaction the suspension is cooled to room temperature, the precipitate is separated by centrifugation at 10,000 g for 10 minutes, washed with deionized water and ethanol and dried at 60°C for 24 hours, the resulting β-FeOOH akagenite particles are dispersed in 20 ml of ethylene glycol using ultrasonic treatment with a power of 100 W for 15 minutes, the resulting suspension is placed in an autoclave and subjected to hydrothermal treatment at 200°C for 8 hours, after recovery the color of the particles changes from orange to black, which indicates formation of the magnetite phase Fe3O4, the finished particles of the rod form of magnetite are separated by a magnet,washed successively with water and ethanol and dried at 60°C for 24 hours, then a SiO2 shell is formed, a 50 mg sample of dried magnetite rods is dispersed in 5 ml of deionized water, dispersion is carried out for five cycles, each of which includes stirring at 300 rpm for 10 min and ultrasonic treatment with a power of 45 W for 3 min, 100% isopropanol is added to the resulting suspension at the rate of 15 ml of isopropanol for every 5 ml of water, after which the mixture is stirred for 20 min at 300 rpm, then, while stirring, 0.5 ml of a 25% NH4OH solution and 0.5 ml of tetraethoxysilane are introduced into the water-alcohol dispersion of particles for every 50 mg of rods, the reaction of the SiO2 shell formation is carried out at room temperature for 20 hours with constant stirring, at the end of the incubation of the particles cleaned by magnetic decantation with deionized water at least five times, the process of forming a silicon dioxide shell is repeated twice,purified rod-shaped particles of Fe3O4@SiO2 are dispersed in a 5% solution of polyethylene glycol with a molecular weight of 4000 Da for additional stabilization of the suspension.

2. A method for producing rod-shaped magnetite particles for isolating DNA from whole blood according to claim 1, characterized in that the rod-shaped particles with a formed first shell of silicon dioxide Fe3O4@SiO2 are dispersed in a water-alcohol solution, then 0.7 ml of acetic acid and 0.5 ml of N-[3-(trimethoxysilyl)propyl]ethylenediamine are added for every 50 mg of particles, incubation is carried out for 20 hours at 300 rpm, then the Fe3O4@SiO2–NH2 particles are purified by magnetic decantation and dispersed in a 5% solution of polyethylene glycol with a molecular weight of 4000 Da.

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