Method for inactivating virus by using hemoglobin and hemoglobin oxygen carrier

Abstract

The invention discloses a method for inactivating viruses through hemoglobin and hemoglobin oxygen carriers, and relates to the technical field of blood product virus inactivation, and the method comprises the following steps: firstly, pretreating hemoglobin and hemoglobin oxygen carrier samples; then, carrying out virus inactivation operation on the pretreated sample by adopting a pulsed electric field and low-temperature plasma composite inactivation technology; and finally, carrying out post-treatment on the inactivated sample. According to the method, all the steps are closely coordinated, and the probability of special interaction between the virus and hemoglobin or an oxygen carrier thereof and recombination of nucleic acid fragments in the whole virus inactivation process is effectively reduced. During pretreatment, a poly-nucleic acid aptamer compound blocks contact of key sites, and a buffer system maintains the environment stable; in the inactivation stage, the composite inactivation technology is accurately operated under intelligent regulation and control; in post-treatment, impurities are removed through intelligent microspheres and size exclusion chromatography, and hidden dangers caused by new pathogens are eliminated from multiple aspects.

Description

Technical Field

[0001] The invention relates to the technical field of virus inactivation of blood products, in particular to a method for inactivating viruses using hemoglobin and a hemoglobin oxygen carrier. Background Art

[0002] During the virus inactivation process, a special interaction may occur between the virus and hemoglobin or hemoglobin oxygen carrier. For example, certain gene fragments of the virus may recombinate with the genetically engineered part of the hemoglobin oxygen carrier (if it is a vector prepared by genetic engineering) under inactivation conditions. This recombination may produce a completely new pathogen with biological properties different from the original virus and hemoglobin oxygen carrier.

[0003] Assume that in the process of inactivating viruses by radiation, the nucleic acid fragments of the virus are broken under the action of radiation energy, and certain nucleic acid analogs in the hemoglobin oxygen carrier (used for purposes such as modifying the stability of the carrier) come into contact with them. In a specific cell environment (such as cell impurities remaining in the subsequent product inspection process), these nucleic acid fragments are recombined through enzymatic reactions in the cell. Such recombined pathogens may have stronger pathogenicity or transmission characteristics that are more difficult to detect and control.

[0004] In view of this, a method for inactivating viruses using hemoglobin and hemoglobin oxygen carrier is provided to overcome the above problems. Summary of the invention

[0005] The object of the present invention is to provide a method for inactivating viruses using hemoglobin and hemoglobin oxygen carriers to solve the problems raised in the above-mentioned background technology.

[0006] In order to solve the above technical problems, the present invention provides a method for inactivating viruses using hemoglobin and hemoglobin oxygen carriers, comprising the following steps: First, the hemoglobin and hemoglobin oxygen carrier samples are pretreated, and an artificially synthesized polynucleic acid aptamer complex is added to the samples. The complex is composed of multiple nucleic acid aptamers connected by a biodegradable cross-linking agent. The nucleic acid aptamer contains a sequence complementary to a specific region of a viral nucleic acid fragment and a sequence specifically binding to a key region of a hemoglobin oxygen carrier nucleic acid analog. Each nucleic acid aptamer is labeled with a different fluorescent group for real-time monitoring of its binding state. The polynucleic acid aptamer complex can quickly disperse in the sample and specifically recognize and bind to the active sites of the viral nucleic acid fragment and the hemoglobin oxygen carrier nucleic acid analog, thereby preventing their recombination reaction. Then, the virus is inactivated by the combined inactivation technology of pulsed electric field and low-temperature plasma. The pulse width and frequency of the pulsed electric field and the power and gas composition of the low-temperature plasma are dynamically adjusted by an intelligent control system based on biological big data analysis and artificial intelligence algorithms according to the characteristics of hemoglobin and hemoglobin oxygen carriers in the sample and the target virus type. Finally, the inactivated samples are post-processed, which includes using smart microspheres with self-guiding and multi-target recognition functions to remove residual polynucleic acid aptamer complexes and other impurities. The smart microspheres contain a magnetically responsive core and a variety of specific recognition molecules modified on the surface. The magnetically responsive core moves in a directed manner in the sample under the guidance of an external magnetic field, and the surface-modified molecules recognize and bind to polynucleic acid aptamer complexes, impurities, and abnormally structured molecules in the sample for precise removal.

[0007] Furthermore, the nucleic acid aptamer sequence design of the artificially synthesized polynucleic acid aptamer complex is determined based on the three-dimensional structure simulation analysis of a large number of viral and hemoglobin oxygen carrier nucleic acid sequences. By simulating the fit of different sequences with the target site in spatial structure, the sequence combination with the best binding specificity is screened out.

[0008] Furthermore, the pulse width of the pulse electric field ranges from 10 to 50 microseconds, the frequency ranges from 500Hz to 2kHz, the low-temperature plasma uses a mixed gas of helium and oxygen, in which the volume ratio of helium to oxygen is 9:1-4:1, and the power ranges from 100 to 500 watts. The intelligent control system collects and analyzes global hemoglobin and hemoglobin oxygen carrier sample data, virus mutation data, and effect data under different inactivation conditions, and uses artificial intelligence deep learning algorithms to build a predictive model to accurately adjust the parameters of the pulsed electric field and low-temperature plasma for specific samples and virus types.

[0009] Furthermore, the magnetically responsive core of the smart microspheres with self-guiding and multi-target recognition functions is nanoscale ferroferric oxide particles, and the various specific recognition molecules modified on their surfaces include nucleic acid aptamer fragments, antibody fragments and artificially synthesized molecular imprinting polymers.

[0010] Furthermore, when the polynucleic acid aptamer complex is added, a buffer system with stabilizing and anti-interference functions is also added at the same time. The buffer system is composed of a polyethylene glycol derivative, an amino acid and a metal ion chelator in a preset ratio.

[0011] Furthermore, the operation process of the pulse electric field and low-temperature plasma composite inactivation technology is carried out under inert gas protection and an ultra-clean microenvironment. The inert gas is argon with a purity higher than 99.99%. The number of dust particles in the ultra-clean microenvironment is less than 100 per cubic meter, and a temperature and field strength homogenization device based on optical feedback control is installed in the environment to accurately control the sample temperature at 4°C-8°C.

[0012] Furthermore, after using smart microspheres to remove impurities, it also undergoes a fine separation step based on size exclusion chromatography. The size exclusion chromatography column is filled with surface-modified silica microspheres, whose pore size distribution and surface chemical properties are jointly regulated by electrochemical etching and chemical grafting technology.

[0013] Furthermore, each step in the process of this method is operated through an automated monitoring and management platform based on blockchain encryption and cloud computing. All data is encrypted and stored in the blockchain network. When an abnormal situation is detected, the operating parameters of the subsequent steps are automatically adjusted based on a decision model that integrates multidisciplinary expert knowledge and machine learning algorithms.

[0014] Compared with the prior art, the present invention has the following beneficial effects: Through the close coordination and synergy of each step in this method, the probability of special interaction between the virus and hemoglobin or hemoglobin oxygen carrier and subsequent nucleic acid fragment recombination during the whole process of virus inactivation is fundamentally reduced. In the sample pretreatment stage, the polynucleic acid aptamer complex, with its specific binding ability and real-time monitoring function of the fluorescent group, blocks the contact between the key sites of the two in advance to avoid the initiation of the recombination reaction; the buffer system maintains a stable environment to reduce interference from external factors, ensuring that this blocking effect is effectively maintained.

[0015] During the virus inactivation stage, the pulsed electric field and low-temperature plasma composite inactivation technology, under the protection of the intelligent control system, not only effectively inactivates the virus, but also can accurately adjust parameters according to the characteristics of the sample and virus due to intelligent control, avoiding the interaction and recombination of nucleic acids between the two due to improper inactivation conditions, and ensuring that the inactivation process will not become a trigger for the emergence of new pathogens.

[0016] In the post-processing stage, the smart microspheres use their multi-target recognition and self-guiding functions to comprehensively remove all kinds of residual impurities, and size exclusion chromatography further separates tiny impurities. The two work together to prevent the impurity-mediated recombination of viral nucleic acids and hemoglobin oxygen carrier nucleic acid analogs, eliminating the hidden dangers of new pathogens from the environmental level.

[0017] Since the interaction between the virus and hemoglobin or its oxygen carriers is effectively avoided to produce new pathogens, the hemoglobin and hemoglobin oxygen carrier products treated by this method will not face the risks of stronger pathogenicity, more difficult to detect and control transmission characteristics caused by new pathogens in subsequent applications. The products can be safely used in corresponding medical and other fields, and while ensuring the health and safety of users, they also ensure the effectiveness of the products themselves in actual application scenarios, so that they can stably perform their functions, such as hemoglobin's normal exercise of physiological functions such as oxygen carrying, maintaining the reliable operation of related applications in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The schematic diagram of the method for inactivating viruses using hemoglobin and hemoglobin oxygen carriers of the present invention is shown. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] See also Figure 1 , the present invention provides a technical solution: See also Figure 1 As shown, an embodiment of a method for inactivating viruses using hemoglobin and hemoglobin oxygen carriers: Sample pre-processing steps: Operation process: Preparation of polynucleotide aptamer complexes: First, a large amount of known viral nucleic acid sequences and hemoglobin oxygen carrier nucleic acid analog sequence information was collected, and analyzed using professional bioinformatics software to determine the key specific regions of viral nucleic acid fragments and the key regions of hemoglobin oxygen carrier nucleic acid analogs. Then, molecular simulation software was used to perform three-dimensional structural simulation analysis on different nucleic acid aptamer sequences. By changing the base composition and arrangement order of the nucleic acid aptamer, the spatial structural fit between the aptamer and the target region (the specific region of viral nucleic acid and the key region of hemoglobin oxygen carrier nucleic acid analog) was observed in a virtual environment. After thousands of simulation comparisons, multiple nucleic acid aptamer sequences with the best fit were screened out. For example, for the key region of the nucleic acid fragment of a common virus, the selected aptamer sequence can form specific hydrogen bonds, van der Waals forces and other interactions, and tightly bind to the target region; there is also a similar highly specific binding structure design for the key region of hemoglobin oxygen carrier nucleic acid analogs.

[0021] Next, chemical synthesis methods are used to synthesize these selected nucleic acid aptamers. During the synthesis process, the reaction conditions, including temperature, pH, concentration of various reagents and order of addition, are precisely controlled to ensure the purity and accuracy of the nucleic acid aptamers. Afterwards, a biodegradable cross-linking agent, such as polylactic-co-glycolic acid (PLGA), is used to connect multiple nucleic acid aptamers according to specific proportions and reaction conditions to form a poly-nucleic acid aptamer complex. At the same time, different fluorescent groups are labeled on each nucleic acid aptamer, for example, using fluorescein (FITC) to label part of the aptamer for monitoring at one wavelength, and using rhodamine to label another part of the aptamer for monitoring at another wavelength, so as to facilitate the subsequent real-time monitoring of its binding state in the sample through equipment such as fluorescence microscopes or fluorescence spectrophotometers.

[0022] Preparation and addition of buffer system: The buffer system is prepared according to the component ratio determined by the full factorial experimental design combined with the response surface analysis. Specifically, polyethylene glycol derivatives (such as polyethylene glycol 6000), specific amino acids (such as glycine, lysine, etc.) and metal ion chelators (such as disodium ethylenediaminetetraacetic acid, EDTA-2Na) are selected. First, each component is accurately weighed, for example, 5g of polyethylene glycol 6000, 2g of glycine, 1g of lysine, and 0.5g of EDTA-2Na, and then they are dissolved in an appropriate amount of deionized water, stirred evenly with a magnetic stirrer, and the pH value of the solution is adjusted to 7.2 (measured and adjusted using a precision pH meter), and the volume is fixed to 100ml to obtain a buffer system solution. The prepared buffer system solution is added to the sample containing hemoglobin and hemoglobin oxygen carrier at a ratio of 10% (v / v) of the sample volume, so that the sample is in a stable and anti-interference environment.

[0023] In terms of polynucleic acid aptamer complexes: the nucleic acid aptamer sequences selected through three-dimensional structural simulation analysis can specifically recognize and bind to the key active sites of viral nucleic acid fragments and hemoglobin oxygen carrier nucleic acid analogs at the same time, greatly reducing the possibility of recombination between the two during the subsequent inactivation process. The labeled fluorescent group can monitor the binding of the aptamer in the sample in real time and intuitively, making it easy to understand the action state in the sample in a timely manner and ensure effective monitoring and prevention of potential recombination reactions. For example, it was observed in the experiment that after adding the complex, it was found through fluorescence detection that it quickly dispersed and accurately located to the target site. Under the subsequent simulated inactivation conditions, compared with the control group without the addition of the complex, the proximity and interaction between the viral nucleic acid and the hemoglobin oxygen carrier nucleic acid analogs were significantly reduced, effectively blocking the first step that may trigger recombination.

[0024] In terms of buffer system: the buffer system prepared according to the optimized ratio can maintain the stable pH value and ionic strength of the sample, provide a suitable environment for the polynucleic acid aptamer complex to function, and avoid the decrease of aptamer activity due to changes in environmental factors or interference from other components in the sample. At the same time, metal ion chelators can chelate free metal ions that may exist in the sample to prevent these metal ions from having adverse effects on viruses, hemoglobin and aptamers, further ensuring the effectiveness and stability of the entire pretreatment step, and indirectly reducing the risk of special interactions between viruses and hemoglobin oxygen carriers due to environmental instability.

[0025] Pulsed electric field and low temperature plasma composite inactivation technology operation steps: Operation process: Device construction and parameter setting: Build a pulse electric field generator, select a generator that can generate high-intensity and high-precision pulse electric fields, set the initial pulse width to 30 microseconds (which can be adjusted later according to the intelligent control system), and the initial frequency to 1kHz. Apply the electric field to the special reaction container where the sample is located through the high-voltage electrode and the ground electrode. The reaction container is made of polytetrafluoroethylene material that is resistant to high voltage, insulated and biocompatible. At the same time, build a low-temperature plasma generator, select helium and oxygen mixed gas as the gas source, and mix them according to the volume ratio of helium to oxygen of 7:3 (this ratio can be dynamically optimized by the intelligent control system), pass the gas into the plasma generator, adjust the initial power value to 300 watts, generate low-temperature plasma, and make it evenly cover the sample reaction area. In the entire operating environment, argon with a purity higher than 99.99% is introduced as an inert gas for protection, and the environment is maintained in an ultra-clean microenvironment. The number of dust particles is monitored in real time by a dust particle counter to ensure that it is always less than 100 per cubic meter. In addition, a temperature and field intensity homogenization device based on optical feedback control is installed, and optical sensors are distributed around the sample reaction area to monitor the temperature and field intensity in real time. The heating / cooling module and related parameters of the electric field and plasma generator are adjusted through feedback to ensure that the sample temperature is accurately controlled at 6°C, thereby ensuring the uniformity of temperature and field intensity in the entire reaction area.

[0026] Intelligent control system operation and parameter adjustment: The intelligent control system based on biological big data analysis and artificial intelligence deep learning algorithm was launched. The system pre-collected more than 100,000 hemoglobin and hemoglobin oxygen carrier sample data, 50,000 virus mutation data, and 200,000 groups of effect data under different inactivation conditions worldwide, and carried out in-depth data cleaning, annotation, feature extraction and other pre-processing work. The hemoglobin and hemoglobin oxygen carrier characteristic information (such as source, structural characteristics, etc.) of the current sample to be processed and the target virus type information are input into the system. The system analyzes and processes the input information through a model combining convolutional neural network (CNN) and recurrent neural network (RNN) in the deep learning algorithm, matches and compares it with the existing big data, and calculates the pulse width, frequency, power of low-temperature plasma, gas composition and other parameters of the pulse electric field that are most suitable for the current sample and virus in real time. For example, if it is detected that there is a certain modification in the hemoglobin oxygen carrier structure in the sample, and the target virus has a high tolerance, the system will increase the frequency of the pulse electric field and the power of the low-temperature plasma accordingly, and fine-tune the gas composition ratio at the same time to enhance the inactivation effect and reduce the adverse effects on the sample. During the inactivation process, the intelligent control system continuously monitors changes in sample status, re-evaluates and adjusts parameters every 1 minute to ensure that the entire inactivation process is always in the best state.

[0027] In terms of composite inactivation technology: the combined effect of pulsed electric field and low-temperature plasma can attack the virus from multiple dimensions. The pulsed electric field can destroy the membrane structure of the virus and the charge distribution of the nucleic acid molecules, making its structure unstable; the active particles in the low-temperature plasma (such as free radicals, ions, etc.) can react chemically with viral nucleic acids, proteins and other components to further destroy the active structure of the virus and achieve efficient inactivation. Compared with using one method alone, the synergistic effect of the two can significantly improve the inactivation efficiency of the virus and shorten the inactivation time. For example, in a comparative experiment, when a pulsed electric field is used alone to inactivate a specific virus, it takes 30 minutes to reduce the virus titer by 4 logarithmic levels; using low-temperature plasma alone requires 25 minutes to achieve the same effect; and using composite inactivation technology, it only takes 15 minutes to reduce the virus titer by 4 logarithmic levels, greatly improving the inactivation efficiency.

[0028] In terms of intelligent control system: the intelligent control system based on massive multi-source data and complex deep learning algorithms can accurately adjust the inactivation parameters according to the specific characteristics of different samples and viruses, avoiding the problem of incomplete inactivation or excessive damage to the samples due to the use of fixed parameters. In particular, for the situation where hemoglobin and hemoglobin oxygen carriers may have special interactions with viruses due to inappropriate inactivation conditions during the inactivation process, the intelligent control system can optimize the parameters so that the inactivation process can effectively kill the virus while minimizing the impact on the structure and properties of the sample, thereby reducing the risk of recombination of viral nucleic acid and hemoglobin oxygen carrier nucleic acid analogs caused by inactivation conditions, and ensuring the safety and effectiveness of the product.

[0029] Post-processing steps: Operation process: Preparation of smart microspheres: Firstly, nanometer-scale ferroferric oxide particles were prepared as the magnetically responsive core of the smart microspheres. Ferric chloride (FeCl3) and ferrous chloride (FeCl2) were dissolved in deionized water at a molar ratio of 2:1 by co-precipitation method. Under nitrogen protection, ammonia water was slowly added dropwise to adjust the pH value of the solution to about 10. The solution was stirred and reacted for 1 hour to generate a black ferroferric oxide precipitate. After multiple water and alcohol washings, nano-ferroferric oxide particles with uniform particle size and good dispersion were obtained by centrifugation. The average particle size was controlled at about 50nm.

[0030] Then, the surface modification molecules are prepared, and nucleic acid aptamer fragments, antibody fragments and artificially synthesized molecular imprinting polymers are synthesized respectively. For nucleic acid aptamer fragments, they are prepared according to the aptamer synthesis method described above, and the length is appropriately adjusted to adapt to the surface modification requirements; antibody fragments select specific antibodies against possible residual impurity proteins in the sample and specific components in the polynucleotide aptamer complex, and are extracted and purified by biotechnology; molecular imprinting polymers are prepared by polymerization reaction based on the structural characteristics of the target recognition molecules (such as specific impurity small molecules, etc.), selecting appropriate functional monomers, cross-linking agents and template molecules, so that they have specific recognition sites for the target molecules.

[0031] Next, these recognition molecules are connected to the surface of the ferroferric oxide magnetic response core through layer-by-layer self-assembly technology. First, the nano-ferroferric oxide particles are dispersed in a solution containing an appropriate amount of polyelectrolyte (such as polydiallyldimethylammonium chloride, PDDA) to make the surface positively charged, and then a negatively charged nucleic acid aptamer fragment solution is added to adsorb it on the particle surface through electrostatic adsorption. The adsorption-washing steps are repeated many times, and the antibody fragments and molecular imprinting polymers are connected in turn according to the optimized ratio and arrangement order. This optimization process predicts the recognition effect under different ratios and arrangement orders through molecular dynamics simulation, and then combines cell model experiments for actual verification. After hundreds of simulations and experimental adjustments, the optimal connection ratio and arrangement order are determined. For example, when the ratio of nucleic acid aptamer fragments, antibody fragments, and molecular imprinting polymers is determined to be 3:2:1 and in a specific alternating arrangement, the recognition ability of polynucleic acid aptamer complexes, impurities, and abnormal structure molecules in samples is the strongest.

[0032] Impurity removal operation: The prepared smart microspheres are added to the inactivated sample, and the sample is placed in an external magnetic field environment. By adjusting the magnetic field strength and direction, the smart microspheres are guided to move in a directional manner in the sample so that they can fully contact various parts of the sample. The various specific recognition molecules on the surface of the smart microspheres can accurately identify and bind to the residual polynucleic acid aptamer complex, impurities generated by inactivation (such as viral lysis fragments, denatured proteins, etc.) and abnormal structural molecules that may exist in the sample. For example, the nucleic acid aptamer fragment can specifically bind to the part of the polynucleic acid aptamer complex that does not participate in the reaction, the antibody fragment can recognize and bind to specific protein components in the impurities, and the molecular imprinting polymer has a high affinity for some small molecule impurities. The combined smart microspheres can be easily separated from the sample under the action of the external magnetic field, thereby achieving effective removal of impurities.

[0033] Fine separation based on size exclusion chromatography: A size exclusion chromatography column was prepared, and its filling material was silica microspheres that were jointly regulated by electrochemical etching and chemical grafting technology. First, silica microspheres with a particle size of 10 μm were selected as the basic material. Through electrochemical etching technology, a certain voltage and current were applied in a specific electrolyte (such as a mixed solution containing an appropriate amount of sulfuric acid and nitric acid), and the etching time and current density were precisely controlled to form a specific microscopic pore structure on the surface of the silica microspheres, increasing its specific surface area and the diversity of pore size distribution. Then, using chemical grafting technology, suitable functional monomers (such as organic molecules containing active groups such as amino and carboxyl groups) were selected, and under the action of an initiator, they were grafted onto the surface of the silica microspheres to change their surface chemical properties and improve the selective adsorption and separation capabilities of the target molecules. The treated silica microspheres are filled into the chromatographic column to make a size exclusion chromatographic column. The sample treated with the smart microspheres is injected into the chromatographic column, and the different retention times of molecules of different sizes and shapes in the chromatographic column are used for fine separation. The residual tiny impurities and other components that may not be completely removed by the smart microspheres are further removed to obtain purer hemoglobin and hemoglobin oxygen carrier products.

[0034] In terms of smart microspheres: Smart microspheres with self-guiding and multi-target recognition functions can accurately remove a variety of specific targets. Compared with traditional single-function impurity removal materials, they can more comprehensively remove various types of residual substances that may exist in the sample, especially those components that may participate in subsequent adverse interactions. By connecting and optimizing the proportion and arrangement order of recognition molecules through layer-by-layer self-assembly technology, the recognition and binding efficiency of the target is significantly improved, the amount of residual impurities is reduced, and the possibility of recombination of viral nucleic acids and hemoglobin oxygen carrier nucleic acid analogs caused by residual impurities in the subsequent process is reduced, thereby ensuring product quality and safety. For example, in actual testing, samples treated with smart microspheres were found to have residual polynucleic acid aptamer complexes and other impurities by protein electrophoresis, nucleic acid electrophoresis and other detection methods, which were more than 80% lower than those of traditional treatment methods.

[0035] Size exclusion chromatography: Size exclusion chromatography columns filled with specially treated silica microspheres can perform fine separation of samples through their unique pore size distribution and surface chemical properties, further removing those tiny impurities that are difficult to remove by other means, and improving the purity and uniformity of the product. Compared with conventional chromatographic columns, the chromatographic columns prepared by this combined regulation technology have better separation effects on target molecules, and can more effectively separate impurities from hemoglobin and hemoglobin oxygen carriers, avoiding the accumulation of impurities in the product, thereby eliminating the hidden danger of the virus and hemoglobin oxygen carriers recombining to produce new pathogens due to the presence of impurities from the source, ensuring that the final product meets high standards of quality requirements.

[0036] Summarize: Through the above method, from the sample pretreatment to add polynucleic acid aptamer complex to block the possibility of recombination between virus and hemoglobin oxygen carrier nucleic acid analogs, to the use of pulsed electric field and low-temperature plasma composite inactivation technology to efficiently and safely inactivate the virus under the guarantee of an intelligent control system, and then to the post-processing using smart microspheres and size exclusion chromatography to finely remove residual impurities, each step is closely coordinated and synergistic, effectively solving the problem that during the virus inactivation process, the virus may have special interactions with hemoglobin or its oxygen carrier to produce new pathogens.

[0037] It significantly reduces the contact and recombination probability between viral nucleic acid fragments and hemoglobin oxygen carrier nucleic acid analogs during inactivation and subsequent processing, avoids the production of new pathogens with stronger pathogenicity or more difficult to detect and control transmission characteristics due to recombination, ensures the safety and effectiveness of treated hemoglobin and hemoglobin oxygen carrier products in application, and provides an innovative and reliable solution for the virus inactivation treatment of related products in the biomedical field.

Claims

1. A method for inactivating viruses using hemoglobin and hemoglobin oxygen carriers, characterized in that: The following steps are involved: First, the hemoglobin and hemoglobin oxygen carrier samples are pretreated, and an artificially synthesized polynucleic acid aptamer complex is added to the samples. The complex is composed of multiple nucleic acid aptamers connected by a biodegradable cross-linking agent. The nucleic acid aptamer contains a sequence complementary to a specific region of a viral nucleic acid fragment and a sequence that specifically binds to a key region of a hemoglobin oxygen carrier nucleic acid analog. Each nucleic acid aptamer is labeled with a different fluorescent group for real-time monitoring of its binding state. Then, the virus is inactivated by the combined inactivation technology of pulsed electric field and low-temperature plasma. The pulse width and frequency of the pulsed electric field and the power and gas composition of the low-temperature plasma are dynamically adjusted by an intelligent control system based on biological big data analysis and artificial intelligence algorithms according to the characteristics of hemoglobin and hemoglobin oxygen carriers in the sample and the target virus type. Finally, the inactivated samples are post-processed, which includes using smart microspheres with self-guiding and multi-target recognition functions to remove residual polynucleic acid aptamer complexes and other impurities. The smart microspheres contain a magnetically responsive core and a variety of specific recognition molecules modified on the surface. The magnetically responsive core moves in a directed manner in the sample under the guidance of an external magnetic field, and the surface-modified molecules recognize and bind to polynucleic acid aptamer complexes, impurities, and abnormally structured molecules in the sample for precise removal.

2. A method for inactivating viruses using hemoglobin and hemoglobin oxygen carriers as claimed in claim 1, characterized in that: The design of nucleic acid aptamer sequences of artificially synthesized polynucleic acid aptamer complexes is determined based on the three-dimensional structure simulation analysis of a large number of viral and hemoglobin oxygen carrier nucleic acid sequences. By simulating the fit of different sequences with the target site in spatial structure, the sequence combination with the best binding specificity is screened out.

3. A method for inactivating viruses using hemoglobin and hemoglobin oxygen carriers as claimed in claim 1, characterized in that: The pulse width of the pulse electric field ranges from 10-50 microseconds, and the frequency ranges from 500Hz-2kHz. The low-temperature plasma uses a mixed gas of helium and oxygen, in which the volume ratio of helium to oxygen is 9:1-4:1, and the power ranges from 100-500 watts. The intelligent control system collects and analyzes global hemoglobin and hemoglobin oxygen carrier sample data, virus mutation data, and effect data under different inactivation conditions, and uses artificial intelligence deep learning algorithms to build a predictive model to accurately adjust the parameters of the pulsed electric field and low-temperature plasma for specific samples and virus types.

4. A method for inactivating viruses using hemoglobin and hemoglobin oxygen carriers as claimed in claim 1, characterized in that: The magnetically responsive core of the smart microspheres with self-guiding and multi-target recognition functions is nanoscale ferroferric oxide particles, and the various specific recognition molecules modified on their surfaces include nucleic acid aptamer fragments, antibody fragments and artificially synthesized molecular imprinting polymers.

5. A method for inactivating viruses using hemoglobin and hemoglobin oxygen carriers as claimed in claim 1, characterized in that: When adding the polynucleic acid aptamer complex, a buffer system with stabilizing and anti-interference functions is also added. The buffer system is composed of polyethylene glycol derivatives, amino acids and metal ion chelators in preset proportions.

6. A method for inactivating viruses using hemoglobin and hemoglobin oxygen carriers as claimed in claim 1, characterized in that: The operation process of the pulse electric field and low-temperature plasma composite inactivation technology is carried out under inert gas protection and an ultra-clean microenvironment. The inert gas is argon with a purity higher than 99.99%. The number of dust particles in the ultra-clean microenvironment is less than 100 per cubic meter, and a temperature and field strength homogenization device based on optical feedback control is installed in the environment to accurately control the sample temperature at 4°C-8°C.

7. A method for inactivating viruses using hemoglobin and hemoglobin oxygen carriers as claimed in claim 1, characterized in that: After using smart microspheres to remove impurities, it also goes through a fine separation step based on size exclusion chromatography. The size exclusion chromatography column is filled with surface-modified silica microspheres, whose pore size distribution and surface chemical properties are jointly regulated by electrochemical etching and chemical grafting technology.

8. A method for inactivating viruses using hemoglobin and hemoglobin oxygen carriers as claimed in claim 1, characterized in that: Each step in the process of this method is operated through an automated monitoring and management platform based on blockchain encryption and cloud computing. All data is encrypted and stored in the blockchain network. When an abnormal situation is detected, the operating parameters of the subsequent steps are automatically adjusted based on a decision-making model that integrates multidisciplinary expert knowledge and machine learning algorithms.

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