Preparation method of high-stability manual buffer solution

Abstract

The invention discloses a preparation method of a high-stability manual buffer solution, which comprises the following steps of: introducing two types of slow-release porous particle structures, namely a first type of particles loaded with acidic or alkaline components and a second type of particles loaded with silver nanoparticles, so that continuous slow release and dynamic adjustment of the components in the buffer solution are realized; the pH drift and the aging of a buffer system are effectively delayed; meanwhile, slow release of the silver nanoparticles endows the buffer solution with lasting antibacterial protection, microbial growth is remarkably inhibited, and due to the structural design, the long-term pH stability and storage safety of the buffer solution are remarkably improved, the clarity and physicochemical properties of the buffer solution are guaranteed, and the stability of the buffer solution is improved. The method greatly expands the application range in the fields of high-requirement biological experiments, medical detection, long-term sample storage and the like, the particle addition amount and the component proportion can be flexibly adjusted according to actual requirements, the method adapts to different pH ranges and application scenes, and various experiment requirements of protein purification, long-term sample storage, high-sensitivity analysis and detection are met.

Description

Technical Field

[0001] The present invention relates to the technical field of buffer solution preparation, and more particularly to a method for preparing a manual buffer solution with high stability. Background Art

[0002] At present, buffer solutions, as important reagents for basic experiments and applications in biology, medicine, analytical chemistry and other fields, have increasingly higher requirements for their pH stability and storage cycle. Conventional buffer solutions mostly use traditional component mixing methods, and are used directly after adjusting the acidic and alkaline components to the target pH. This type of buffer solution is simple to prepare and widely used, and is the basis for routine laboratory operations.

[0003] However, existing traditional buffer solutions are prone to problems such as pH drift, microbial contamination, and decreased buffering capacity during actual storage and use. Especially during long-term storage or repeated use, buffer solutions are affected by environmental factors (such as carbon dioxide absorption, microbial growth, component degradation, etc.), which often lead to pH fluctuations, decreased clarity, or precipitation, thereby affecting the accuracy of the experiment and the reliability of the results. In addition, conventional buffer solutions are difficult to balance long-term pH stability and antibacterial ability, and cannot meet the needs of high-standard experiments and sample preservation.

[0004] Therefore, in view of the above technical problems, it is necessary to provide a method for preparing a manual buffer solution with high stability. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing a high-stability manual buffer solution to solve the above-mentioned problems.

[0006] In order to achieve the above-mentioned purpose, the technical solution provided by one embodiment of the present invention is as follows:

[0007] A method for preparing a high-stability manual buffer solution comprises the following steps:

[0008] S1: Select acidic and alkaline components based on the pH range and purpose of the target buffer;

[0009] S2: Accurately weighing the acidic component and the alkaline component and dissolving them in deionized water so that the initial concentrations of the acidic component and the alkaline component are controlled between 0.1 mol / L and 1 mol / L to prepare an acidic solution and an alkaline solution, respectively;

[0010] S3: At a constant temperature of 20°C to 25°C, add the acidic solution dropwise to the alkaline solution at a rate of 0.5 mL to 1 mL per minute. Simultaneously, use a high-precision pH meter to monitor the pH of the mixed solution in real time and slowly adjust the pH to the target value to ensure that there is no overshoot or drastic fluctuation.

[0011] S4: Filter the prepared buffer solution through a sterile filter with a pore size of 0.22 μm to remove particulate impurities and microorganisms;

[0012] S5: Adding the slow-release porous particles prepared in steps and physically mixed to the buffer solution, wherein the slow-release porous particles comprise:

[0013] The first type of particles are loaded with acidic or alkaline components for sustained release to adjust pH;

[0014] The second type of particles, loaded with silver nanoparticles, are used to inhibit microbial growth in buffer;

[0015] The two types of particles are prepared separately before use and then physically mixed, and then a buffer solution is added together to achieve long-term pH stability and antibacterial properties;

[0016] S6: Dispense the prepared buffer solution into pre-sterilized containers, seal them, and store them at 2°C to 8°C.

[0017] As a further improvement of the present invention, the acidic component in S1 is sodium dihydrogen phosphate, acetic acid or citric acid, and the alkaline component is disodium hydrogen phosphate, tris(hydroxymethyl)aminomethane or sodium bicarbonate.

[0018] As a further improvement of the present invention, the conductivity of the deionized water in S2 is less than 0.5 μS / cm, and a constant temperature magnetic stirrer is used during dissolution, with the stirring speed controlled between 300 rpm and 500 rpm.

[0019] As a further improvement of the present invention, during the real-time pH monitoring process in S3, the electrode of the pH meter needs to undergo a two-point calibration with pH 4.01 and pH 7.00 standard buffer solutions, and the calibration error does not exceed ±0.02.

[0020] As a further improvement of the present invention, the buffer solution in S4 needs to be initially filtered using a filter with a pore size of 0.45 μm before filtering to remove larger particles, and then finely filtered using a sterile filter with a pore size of 0.22 μm.

[0021] As a further improvement of the present invention, the sustained-release porous particles in S5 are silica gel microspheres, zeolite particles or hydroxyapatite particles.

[0022] As a further improvement of the present invention, the method for preparing the first type of particles in S5 comprises the following steps:

[0023] S51: Select a porous material, wash it with deionized water or ethanol, and dry it for later use;

[0024] S52: soaking the porous material in an acidic component solution or an alkaline component solution at room temperature for 4 to 24 hours;

[0025] S53: The porous material after the impregnation is taken out, and the excess solution on the surface is removed, and the porous material is dried by low-temperature vacuum drying or constant temperature drying to obtain sustained-release particles loaded with acidic components or alkaline components.

[0026] As a further improvement of the present invention, in S5, the preparation method of the second type of particles includes:

[0027] S54: Select a porous material, wash it with deionized water or ethanol, and dry it for later use;

[0028] S55: dispersing the porous material in a silver salt solution, wherein the silver salt is preferably silver nitrate with a concentration of 1 to 10 mmol / L;

[0029] S56: adding a reducing agent to the mixed solution under stirring or ultrasonic conditions, wherein the reducing agent is sodium citrate, ascorbic acid or glucose, and reacting at room temperature for 0.5 to 2 hours to reduce the silver ions in situ on the surface and in the pores of the particles to form silver nanoparticles;

[0030] S57: After the reaction is completed, the mixture is filtered, washed with deionized water, and dried at low temperature to obtain sustained-release particles loaded with silver nanoparticles.

[0031] As a further improvement of the present invention, the particle size of the silver nanoparticles in S5 is 1 nm to 100 nm, preferably 10 nm to 30 nm, and the loading amount is 0.01% to 1% of the mass of the sustained-release particles.

[0032] As a further improvement of the present invention, in S5, the total amount of the first and second types of slow-release porous particles added is 0.01% to 1% of the total mass of the buffer solution, and the mass ratio of the first type of slow-release porous particles to the second type of slow-release porous particles is 10:1 to 1:10.

[0033] Compared with the prior art, the advantages of the present invention are:

[0034] (1) This scheme significantly improves the overall performance of the buffer solution by introducing two types of slow-release porous particle structures into the buffer solution. First, the first type of slow-release particles loaded with acidic or alkaline components can achieve continuous and uniform release of the buffer components, so that the buffer system can automatically adjust the pH during long-term storage or multiple uses, greatly slowing down the pH drift caused by external carbon dioxide absorption, component volatilization or degradation, and effectively extending the service life and stability of the buffer solution. Secondly, the second type of slow-release particles loaded with silver nanoparticles slowly release highly effective antibacterial components in the buffer solution, which can permanently inhibit the reproduction and contamination of microorganisms such as bacteria and fungi, ensuring that the buffer solution remains sterile for a long time under room temperature or refrigerated conditions, and reducing the risk of failure of experimental samples due to microbial contamination.

[0035] (2) This scheme uses inorganic materials with stable structure and large specific surface area as carriers for both types of porous particles, such as silica gel, zeolite or hydroxyapatite. This not only improves the loading capacity and release uniformity of functional components, but also greatly enhances the compatibility and dispersibility of particles with buffer solutions, avoiding the generation of turbidity and precipitation. The amount of particles added and the proportion of components can be flexibly adjusted according to actual needs, adapting to different pH ranges and application scenarios, and meeting various experimental needs such as protein purification, long-term sample storage, and high-sensitivity analysis and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the manual buffer preparation method of the present invention;

[0037] Figure 2 This is a schematic diagram of the preparation data of the high-stability buffer solution in an alkaline environment of the present invention;

[0038] Figure 3 This is a schematic diagram of the data structure of the preparation of a high-stability buffer solution in an acidic environment according to the present invention. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention; it is obvious that the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0040] Example 1:

[0041] See also Figure 1 , a method for preparing a high-stability manual buffer solution, comprising the following steps:

[0042] S1: Select acidic and alkaline components based on the pH range and purpose of the target buffer;

[0043] S2: Accurately weighing the acidic component and the alkaline component and dissolving them in deionized water so that the initial concentrations of the acidic component and the alkaline component are controlled between 0.1 mol / L and 1 mol / L to prepare an acidic solution and an alkaline solution, respectively;

[0044] S3: At a constant temperature of 20°C to 25°C, add the acidic solution dropwise to the alkaline solution at a rate of 0.5 mL to 1 mL per minute. Simultaneously, use a high-precision pH meter to monitor the pH of the mixed solution in real time and slowly adjust the pH to the target value to ensure that there is no overshoot or drastic fluctuation.

[0045] S4: Filter the prepared buffer solution through a sterile filter with a pore size of 0.22 μm to remove particulate impurities and microorganisms;

[0046] S5: Adding the sustained-release porous particles prepared in steps and physically mixed to the buffer solution, the sustained-release porous particles comprising:

[0047] The first type of particles are loaded with acidic or alkaline components for sustained release to adjust pH;

[0048] The second type of particles, loaded with silver nanoparticles, are used to inhibit microbial growth in buffer;

[0049] The two types of particles are prepared separately before use and then physically mixed, and then buffer is added together to achieve long-term pH stability and antibacterial properties;

[0050] S6: Dispense the prepared buffer solution into pre-sterilized containers, seal them, and store them at 2°C to 8°C.

[0051] By introducing two types of slow-release porous particle structures—a first type of particle loaded with acidic or alkaline components and a second type of particle loaded with silver nanoparticles—the team achieved sustained slow-release and dynamic regulation of the buffer components, effectively slowing pH drift and buffer system aging. Furthermore, the slow release of the silver nanoparticles imparts a lasting antimicrobial protection to the buffer, significantly inhibiting microbial growth. This structural design not only significantly improves the buffer's long-term pH stability and storage safety, but also ensures its clarity and physicochemical properties, greatly expanding its application in demanding biological experiments, medical testing, and long-term sample storage.

[0052] The acidic component in S1 is sodium dihydrogen phosphate, acetic acid or citric acid, and the alkaline component is disodium hydrogen phosphate, tris(hydroxymethyl)aminomethane or sodium bicarbonate.

[0053] Among them, in the scheme of the present invention, the acidic component is preferably limited to sodium dihydrogen phosphate, acetic acid or citric acid, and the alkaline component is limited to disodium hydrogen phosphate, tris(hydroxymethyl)aminomethane or sodium bicarbonate, which have stable physical and chemical properties, high safety, and are easy to obtain and store.

[0054] Sodium dihydrogen phosphate and disodium hydrogen phosphate together form the classic phosphate buffer system. Changing the molar ratio of the two can achieve precise adjustment of the buffer solution within a wide pH range (approximately 5.8-8.0). The phosphate buffer system has good chemical stability, is not easily degraded by most biological molecules and enzymes, and has a weak chelating effect on metal ions. Therefore, it is widely used in biology, molecular biology, and clinical testing.

[0055] The acetic acid buffer system composed of acetic acid and its sodium salt is suitable for medium-to-low acidic environments with a pH of 3.6-5.6. It can effectively alleviate pH fluctuations caused by external acid-base disturbances. Its molecular structure is simple, its volatility is low, and its compatibility with various organic and inorganic components is good, making it suitable for the preservation and analysis of biological samples.

[0056] Citric acid, as a polycarboxylic acid, can form a buffer system with its sodium salt covering a pH range of 3.0-6.2. Citric acid buffers have excellent antioxidant and chelating properties, effectively preventing catalytic or precipitation reactions with metal ions, thereby improving the long-term stability and applicability of the buffer.

[0057] Tris (hydroxymethylaminomethane) is a key buffer component in molecular biology and protein purification due to its weak alkalinity and high buffering capacity near physiological pH (approximately 7.0-9.0). Its low ionic strength and minimal biointerference provide favorable conditions for precision detection.

[0058] Sodium bicarbonate can form a carbonate buffer system with acidic components, which is suitable for an environment of pH 6.5-8.5. It has strong buffering capacity and is easy to coordinate with other components, which helps to improve the applicability and flexibility of the buffer.

[0059] The conductivity of the deionized water in S2 is less than 0.5 μS / cm, and a constant temperature magnetic stirrer is used during dissolution, with the stirring speed controlled between 300 rpm and 500 rpm.

[0060] Among them, the conductivity of the deionized water used in the step is strictly limited to less than 0.5 μS / cm. Conductivity is a key indicator for measuring the ion content in water. The lower the conductivity, the fewer impurity ions in the water. The use of high-purity deionized water can effectively avoid the interference of inorganic ion impurities on the buffer components, prevent the formation of precipitation or the initiation of side reactions, and ensure the final purity and performance stability of the buffer.

[0061] Especially in highly sensitive biological experiments and analytical tests, trace metal ions and other impurities may affect the pH accuracy and component activity of the buffer. Therefore, low-conductivity deionized water is preferred to help improve the overall quality and long-term storage stability of the buffer.

[0062] In addition, during the component dissolution process, a constant temperature magnetic stirrer is used for dissolution, and the stirring speed is controlled between 300 rpm and 500 rpm. Constant temperature stirring can ensure uniform solution temperature, prevent component decomposition or reduced activity due to local overheating or overcooling, and help accelerate the dissolution process, so that the acidic and alkaline components can be fully dissolved in deionized water.

[0063] The limitation of stirring speed not only ensures the uniformity of solution mixing, but also avoids sputtering loss or bubble entrainment caused by excessive stirring, thereby improving the safety and controllability of experimental operations.

[0064] During the real-time pH monitoring process in S3, the electrode of the pH meter needs to be calibrated at two points using pH 4.01 and pH 7.00 standard buffer solutions, and the calibration error should not exceed ±0.02.

[0065] Among them, in order to ensure higher accuracy and repeatability of pH value control during buffer preparation, the pH meter electrode used for real-time pH monitoring must undergo two-point calibration with pH 4.01 and pH 7.00 standard buffer solutions, and the calibration error must not exceed ±0.02.

[0066] The two-point calibration method uses two standard buffer solutions with known pH values ​​and a wide span to effectively correct the linearity error and zero drift of the pH meter over the entire measuring range, thereby significantly improving the reliability of pH measurements.

[0067] Selecting pH 4.01 and pH 7.00 as calibration points covers both the acidic and neutral regions, adapting to the working pH range of most buffer solutions and improving the representativeness and applicability of the calibration. Especially during the preparation of high-precision buffer solutions, even small pH errors can affect product quality and experimental results. Therefore, limiting the calibration error to ±0.02 helps to minimize the systematic error introduced by the instrument itself and ensure that the pH value of the prepared buffer solution strictly falls within the target range.

[0068] Before filtering the buffer in S4, it is necessary to perform preliminary filtration using a 0.45 μm pore size filter to remove larger particles, and then use a 0.22 μm pore size sterile filter for fine filtration.

[0069] Among them, the filtration process of the buffer solution adopts a two-stage filtration process. First, preliminary filtration is performed through a 0.45μm pore size filter to remove larger particles and suspended impurities in the solution. This step can effectively eliminate undissolved components, dust particles or other large molecular impurities that may be introduced during the preparation process, thereby creating good prerequisites for subsequent precision filtration, avoiding larger particles clogging the precision filter membrane, and improving the efficiency of the filtration operation and the service life of the filter membrane.

[0070] Subsequently, the buffer solution needs to be precisely filtered through a sterile filter with a pore size of 0.22μm. The pore size of 0.22μm can effectively intercept most bacteria and microorganisms, significantly reduce the microbial load of the buffer solution, and further improve the purity and sterility of the buffer solution. This is crucial for ensuring the safety and stability of the buffer solution in long-term storage and biological experiment applications. It can effectively prevent the buffer solution from being contaminated by microorganisms and extend its service life.

[0071] The sustained-release porous particles in S5 are silica gel microspheres, zeolite particles or hydroxyapatite particles.

[0072] Among them, the sustained-release porous particles are preferably silica gel microspheres, zeolite particles or hydroxyapatite particles, all of which have rich pore structures and high specific surface areas, and can achieve efficient loading and slow release of functional components (such as acidic components, alkaline components or silver nanoparticles). Their porous properties not only help to regulate the release rate of the components, but also increase the contact area between the particles and the buffer solution, thereby achieving continuous and stable functional output.

[0073] Silica gel microspheres have good chemical stability and biocompatibility, adjustable surface properties, and can effectively load a variety of active substances and achieve controlled release. Zeolite particles, as natural or synthetic aluminum silicate minerals, have excellent ion exchange capacity and molecular sieve effect, and can be used to load and slowly release a variety of inorganic or organic components. They also have certain adsorption and purification functions. Hydroxyapatite particles are widely used as carrier materials in the biomedicine and environmental fields due to their good compatibility with organisms, adjustable pore structure and surface adsorption capacity. They can stably load and slowly release silver nanoparticles or other functional substances.

[0074] The preparation method of the first type of particles in S5 comprises the following steps:

[0075] S51: Select a porous material, wash it with deionized water or ethanol, and dry it for later use;

[0076] S52: soaking the porous material in an acidic component solution or an alkaline component solution at room temperature for 4 to 24 hours;

[0077] S53: The porous material after the impregnation is taken out, and the excess solution on the surface is removed, and the porous material is dried by low-temperature vacuum drying or constant temperature drying to obtain sustained-release particles loaded with acidic components or alkaline components.

[0078] Among them, the preparation method of the first type of sustained-release porous particles includes key steps such as the selection of porous materials, activation treatment, and loading and drying of functional components. First, through step S51, a porous material with a highly porous structure is selected and thoroughly cleaned with deionized water or ethanol, which can effectively remove impurities and particles on the surface of the material, ensure the purity and uniformity of the subsequent loading process, and thus lay the foundation for the sustained-release performance of the particles.

[0079] In step S52, the cleaned and dried porous material is immersed in an acidic component solution or an alkaline component solution and immersed at room temperature for 4 to 24 hours, so that the active components can fully penetrate and be adsorbed into the pores and surface of the porous material. By reasonably controlling the immersion time and conditions, effective regulation of the component loading and distribution uniformity can be achieved, providing a guarantee for the later sustained-release characteristics of the particles.

[0080] In step S53, the porous material after impregnation is taken out and the excess solution on the surface is removed. Subsequently, it is dried by low-temperature vacuum drying or constant temperature drying to finally obtain sustained-release particles loaded with acidic components or alkaline components. The drying step not only helps to fix the active components and prevent their loss, but also maintains the structural integrity and good dispersibility of the porous material, ensuring that the sustained-release particles have a long-lasting and stable sustained-release effect in the buffer solution.

[0081] Through the above-mentioned optimized preparation process, the first type of sustained-release porous particles with uniform structure, stable load and excellent sustained-release performance can be obtained, thereby achieving continuous adjustment of pH in the buffer solution and effectively improving the long-term stability and reliability of the buffer solution.

[0082] In S5, the preparation method of the second type of particles includes:

[0083] S54: Select a porous material, wash it with deionized water or ethanol, and dry it for later use;

[0084] S55: dispersing the porous material in a silver salt solution, wherein the silver salt is preferably silver nitrate, with a concentration of 1 to 10 mmol / L;

[0085] S56: adding a reducing agent to the mixed solution under stirring or ultrasonic conditions, wherein the reducing agent is sodium citrate, ascorbic acid or glucose, and reacting at room temperature for 0.5 to 2 hours to reduce the silver ions in situ on the surface and in the pores of the particles to form silver nanoparticles;

[0086] S57: After the reaction is completed, the mixture is filtered, washed with deionized water, and dried at low temperature to obtain sustained-release particles loaded with silver nanoparticles.

[0087] Among them, in step S54, a porous material with a good pore structure is first selected, washed with deionized water or ethanol, and then dried for use. This process can remove impurities and potential ionic pollutants on the surface of the material, ensuring that the subsequent loading of silver nanoparticles is more uniform and effective.

[0088] In step S55, the pretreated porous material is dispersed in a silver salt solution, preferably silver nitrate, with a concentration controlled at 1 to 10 mmol / L. Silver ions within this concentration range can fully penetrate and adsorb into the pores and surface of the porous material, providing sufficient precursors for the subsequent reduction reaction.

[0089] In step S56, a reducing agent is added to the mixed solution under stirring or ultrasonic conditions. The reducing agent may be sodium citrate, ascorbic acid, or glucose. The reaction is controlled to proceed at room temperature for 0.5 to 2 hours, thereby reducing the silver ions in situ on the surface and within the pores of the porous particles to form silver nanoparticles with uniform particle size. The reduction reaction is carried out under mild conditions, which ensures the dispersion and stability of the silver nanoparticles while preventing nanoparticle agglomeration or structural damage.

[0090] In step S57, after the reaction is completed, the particles are filtered and then repeatedly washed with deionized water to remove unreacted silver ions and excess reducing agent. Finally, low-temperature drying is used to obtain sustained-release porous particles loaded with silver nanoparticles. Low-temperature drying can effectively retain the structure and activity of the nanoparticles and prevent agglomeration or performance loss caused by high temperature.

[0091] Through the above-mentioned optimized preparation method, nano-scale silver particles can be uniformly loaded on the surface and inside of the porous carrier material, giving the sustained-release particles excellent antibacterial activity and sustained-release properties. This can not only continuously and effectively inhibit the growth of microorganisms in the buffer solution and extend the storage period of the buffer solution, but also ensure the high purity and safety of the buffer solution.

[0092] The particle size of the silver nanoparticles in S5 is 1 nm to 100 nm, preferably 10 nm to 30 nm, and the loading amount is 0.01% to 1% of the mass of the sustained-release particles.

[0093] In S5, the total amount of the first and second types of slow-release porous particles added is 0.01% to 1% of the total mass of the buffer solution, and the mass ratio of the first type of slow-release porous particles to the second type of slow-release porous particles is 10:1 to 1:10.

[0094] Among them, the particle size of silver nanoparticles is limited to 1nm to 100nm, preferably 10nm to 30nm, and its loading amount is 0.01% to 1% of the mass of the sustained-release particles. Silver nanoparticles have broad-spectrum and high-efficiency antibacterial activity. The smaller the particle size, the larger the specific surface area and the higher the contact efficiency with microorganisms, thereby achieving excellent antibacterial effects at lower doses.

[0095] When the particle size is preferably between 10 nm and 30 nm, silver nanoparticles can maintain good dispersibility and stability while effectively penetrating the cell walls of microorganisms, achieving efficient inhibition of a variety of bacteria and fungi. In addition, controlling the loading amount of silver nanoparticles within the range of 0.01% to 1% of the mass of the sustained-release particles can take into account both antibacterial properties and cost control, avoid potential toxicity problems caused by excessive silver release, and help improve the safety of buffer solutions in fields such as biology and medicine.

[0096] At the same time, the present invention limits the total amount of the first and second slow-release porous particles added to 0.01% to 1% of the total mass of the buffer solution, and the mass ratio of the first and second slow-release porous particles is 10:1 to 1:10.

[0097] The ratio design can flexibly adjust the proportion of the two types of particles according to actual needs, thereby optimizing the pH stability and antibacterial properties of the buffer solution. A higher proportion of the first type of sustained-release particles helps to maintain the pH stability of the buffer solution for a long time, while increasing the proportion of the second type of sustained-release particles can significantly enhance the antimicrobial ability of the buffer solution. Strict control of the total addition amount ensures that the clarity and physicochemical properties of the buffer solution will not be affected by the particles, while achieving efficient performance of the functional components, which is beneficial to maintaining the performance of the buffer solution during long-term storage and high-demand applications.

[0098] Through the above-mentioned optimization design, the buffer solution finally obtained can not only achieve long-term and precise control of pH, but also has excellent antibacterial ability and storage stability, greatly expanding its application range in biology, medicine, chemistry and other fields.

[0099] When preparing a high stability buffer in an alkaline environment, the preparation steps include the following conditions:

[0100] 1. Preparation steps

[0101] (1) Goal setting

[0102] Target pH: 8.0.

[0103] Buffer system: disodium hydrogen phosphate (Na2HPO4) / sodium dihydrogen phosphate (NaH2PO4).

[0104] Application: Protein purification.

[0105] (2) Preparation of acid and base component solutions

[0106] Disodium hydrogen phosphate (alkaline component) and sodium dihydrogen phosphate (acidic component) were accurately weighed, with initial concentrations of both being 0.5 mol / L.

[0107] Dissolve in deionized water with a conductivity of <0.5μS / cm, stir with a magnetic stirrer at 300rpm, and maintain a constant temperature of 25°C.

[0108] (3) Dropwise addition and pH adjustment

[0109] Slowly add the sodium dihydrogen phosphate solution (acidic) into the sodium hydrogen phosphate solution (alkaline) at a rate of 0.5 mL / min.

[0110] Real-time pH monitoring, the pH meter is calibrated at pH 4.01 / 7.00 with an error of ≤±0.02.

[0111] Adjust to target pH = 8.0.

[0112] (4) Filtering

[0113] First filter through a 0.45μm filter, and then filter through a 0.22μm sterile filter.

[0114] (5) Preparation and addition of sustained-release porous particles

[0115] The first type of particles: Silica gel microspheres were selected, washed and dried, 70% loaded with sodium dihydrogen phosphate (acidic) and 30% loaded with disodium hydrogen phosphate (alkaline), with a ratio of 7:3, immersed at room temperature for 12 hours, and dried in vacuum at low temperature.

[0116] The second type of particles: silica gel microspheres from the same batch were dispersed in 1.5 mmol / L silver nitrate solution, reduced with ascorbic acid, and reacted for 1 hour. The particle size was about 20 nm and the silver loading was 0.1%.

[0117] Mixing ratio: The mass ratio of the first type of particles to the second type of particles is 4:1.

[0118] Total addition amount: 0.2% of the total mass of the buffer.

[0119] (6) Packaging and storage

[0120] Dispense into glass bottles sterilized at 121°C and store in a refrigerator at 2-8°C.

[0121] in accordance with Figure 2 The data showed that the pH change of the buffer of the present invention was <0.1 after 90 days, which was much better than that of the traditional buffer (>0.3), and there was almost no microbial growth, indicating that the particle sustained-release regulation system significantly extended the effective period of the buffer.

[0122] Example 2:

[0123] The preparation of a highly stable buffer in an acidic environment includes the following steps:

[0124] 1. Preparation steps

[0125] (1) Goal setting

[0126] Target pH: 4.5.

[0127] Buffer system: acetic acid / sodium acetate.

[0128] Purpose: Preservation of biological samples.

[0129] (2) Preparation of acid and base component solutions

[0130] Accurately weigh glacial acetic acid (acidic component) and sodium acetate (alkaline component), with initial concentrations of both being 0.2 mol / L.

[0131] Dissolve in deionized water with a conductivity of <0.5μS / cm, stir magnetically at 500rpm, and maintain a constant temperature of 20℃.

[0132] (3) Dropwise addition and pH adjustment

[0133] Slowly add the sodium acetate solution dropwise to the glacial acetic acid solution at a rate of 1 mL / min.

[0134] Real-time pH monitoring, two-point calibration of pH meter, adjusted to pH = 4.5.

[0135] (4) Filtering

[0136] First filter through a 0.45μm filter and then filter through a 0.22μm sterile filter.

[0137] (5) Preparation and addition of sustained-release porous particles

[0138] The first type of particles: zeolite particles were selected, washed and dried, 85% loaded with glacial acetic acid and 15% loaded with sodium acetate, with a ratio of 17:3, immersed at room temperature for 8 hours, and dried in vacuum at low temperature.

[0139] The second type of particles: the same batch of zeolite was dispersed in 5mmol / L silver nitrate solution, reduced with sodium citrate, and reacted for 2 hours. The particle size was about 15nm and the silver loading was 0.2%.

[0140] Mixing ratio: The mass ratio of the first type of particles to the second type of particles is 2:1.

[0141] Total addition amount: 0.3% of the total mass of the buffer.

[0142] (6) Packaging and storage

[0143] Dispense into sterile plastic tubes and store in a refrigerator at 2-8°C.

[0144] in accordance with Figure 3 The data showed that the pH change of the buffer solution of the present invention was <0.05 after 90 days, which was much better than that of the traditional buffer solution (>0.2), and no colony grew, which significantly improved the storage stability and antibacterial safety of the acidic buffer solution.

[0145] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0146] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for preparing a high-stability manual buffer solution, characterized in that: The following steps are involved: S1: Select acidic and alkaline components based on the pH range and purpose of the target buffer; S2: Accurately weighing the acidic component and the alkaline component and dissolving them in deionized water so that the initial concentrations of the acidic component and the alkaline component are controlled between 0.1 mol / L and 1 mol / L to prepare an acidic solution and an alkaline solution, respectively; S3: At a constant temperature of 20°C to 25°C, add the acidic solution dropwise to the alkaline solution at a rate of 0.5 mL to 1 mL per minute. Simultaneously, use a high-precision pH meter to monitor the pH of the mixed solution in real time and slowly adjust the pH to the target value to ensure that there is no overshoot or drastic fluctuation. S4: Filter the prepared buffer solution through a sterile filter with a pore size of 0.22 μm to remove particulate impurities and microorganisms; S5: Adding the slow-release porous particles prepared in steps and physically mixed to the buffer solution, wherein the slow-release porous particles comprise: The first type of particles are loaded with acidic or alkaline components for sustained release to adjust pH; The second type of particles, loaded with silver nanoparticles, are used to inhibit microbial growth in buffer; The two types of particles are prepared separately before use and then physically mixed, and then buffer is added together to achieve long-term pH stability and antibacterial properties; S6: Dispense the prepared buffer solution into pre-sterilized containers, seal them, and store them at 2°C to 8°C.

2. A method for preparing a high-stability manual buffer according to claim 1, characterized in that: The acidic component in S1 is sodium dihydrogen phosphate, acetic acid or citric acid, and the alkaline component is disodium hydrogen phosphate, tris(hydroxymethyl)aminomethane or sodium bicarbonate.

3. A method for preparing a high-stability manual buffer according to claim 1, characterized in that: The conductivity of the deionized water in S2 is less than 0.5 μS / cm, and a constant temperature magnetic stirrer is used during dissolution, with the stirring speed controlled between 300 rpm and 500 rpm.

4. A method for preparing a high-stability manual buffer according to claim 1, characterized in that: During the real-time pH monitoring process in S3, the electrode of the pH meter needs to undergo a two-point calibration with pH 4.01 and pH 7.00 standard buffer solutions, and the calibration error does not exceed ±0.

02.

5. A method for preparing a high-stability manual buffer according to claim 1, characterized in that: Before filtering the buffer solution in S4, a 0.45 μm pore size filter is used for preliminary filtration to remove larger particles, and then a 0.22 μm pore size sterile filter is used for precision filtration.

6. A method for preparing a high-stability manual buffer according to claim 1, characterized in that: The sustained-release porous particles in S5 are silica gel microspheres, zeolite particles or hydroxyapatite particles.

7. A method for preparing a high-stability manual buffer according to claim 1, characterized in that: The method for preparing the first type of particles in S5 comprises the following steps: S51: Select a porous material, wash it with deionized water or ethanol, and dry it for later use; S52: soaking the porous material in an acidic component solution or an alkaline component solution at room temperature for 4 to 24 hours; S53: The porous material after the impregnation is taken out, and the excess solution on the surface is removed, and the porous material is dried by low-temperature vacuum drying or constant temperature drying to obtain sustained-release particles loaded with acidic components or alkaline components.

8. A method for preparing a high-stability manual buffer according to claim 1, characterized in that: In S5, the preparation method of the second type of particles includes: S54: Select a porous material, wash it with deionized water or ethanol, and dry it for later use; S55: dispersing the porous material in a silver salt solution, wherein the silver salt is preferably silver nitrate with a concentration of 1 to 10 mmol / L; S56: adding a reducing agent to the mixed solution under stirring or ultrasonic conditions, wherein the reducing agent is sodium citrate, ascorbic acid or glucose, and reacting at room temperature for 0.5 to 2 hours to reduce the silver ions in situ on the surface and in the pores of the particles to form silver nanoparticles; S57: After the reaction is completed, the mixture is filtered, washed with deionized water, and dried at low temperature to obtain sustained-release particles loaded with silver nanoparticles.

9. A method for preparing a high-stability manual buffer according to claim 1, characterized in that: The particle size of the silver nanoparticles in S5 is 1 nm to 100 nm, preferably 10 nm to 30 nm, and the loading amount is 0.01% to 1% of the mass of the sustained-release particles.

10. A method for preparing a high-stability manual buffer according to claim 1, characterized in that: In S5, the total amount of the first and second slow-release porous particles added is 0.01% to 1% of the total mass of the buffer solution, and the mass ratio of the first and second slow-release porous particles is 10:1 to 1:10.

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